Investigating novel

methods to reduce
cholesterol level
SMTP Final Research Paper 2016
Ng De Rong Tony (4S3-07) [Leader]
Chay Hui Xiang (4S1-03)
Lai Tian Lang (4S2-08)

Hwa Chong Institution (High School)

Mrs Goh-Yip Cheng Wai

Abstract

An increase in blood cholesterol contributes to cardiovascular diseases, the number

one cause of death worldwide. Statins are currently the most effective in reducing
cholesterol levels and treating patients with high cholesterol. However, these pharmaceutical
agents have been shown to cause several side effects, prompting the need for a more
natural solution to increasing cholesterol levels. Hence, a study was conducted to investigate
the ability of lactic acid bacteria in the removal of cholesterol, explore the mechanism for the
removal of cholesterol by lactic acid bacteria, and examine the effectiveness of kidney beans
and sunflower seeds in inhibiting HMG-CoA reductase in the cholesterol biosynthesis
pathway. Results showed that Lactobacillus plantarum was the most effective in reducing
cholesterol levels and that the mechanism for cholesterol removal included both the binding
to cell wall and active uptake into cells. Sunflower seeds and kidney beans were also shown
to be effective in inhibiting HMG-CoA reductase, with sunflower seeds having 100%
inhibition of the enzyme, similar to pravastatin, a commercial cholesterol reducing drug, and
kidney beans having comparable percentage inhibition of the enzyme compared to
pravastatin.

Introduction

According to a report by the World Health Organisation (WHO), cardiovascular

diseases (CVDs) are the number one cause of death globally, with an estimated 17.5 million
people having died from CVDs in 2012, accounting for 31% of all global deaths. The WHO
has predicted that by 2030, cardiovascular diseases will remain as the leading causes of
death, taking the lives of almost 23.6 million people worldwide. An increased level of blood
cholesterol is a well-known major risk factor for cardiovascular diseases. It has been
reported that hypercholesterolemia contributes to 45% of heart attacks in Western Europe
and 35% of heart attacks in Central and Eastern Europe (Yusuf, Hawken, Ounpuu, Dans,
Avezum, Lanas, McQueen, Budaj, Pais, Varigos and Li, 2004).
Although pharmacological agents such as statins, which inhibit the enzyme HMGCoA reductase can effectively reduce cholesterol levels and treat patients with high
cholesterol, they are known to have severe side effects. Muscle adverse effects are the best
recognized adverse effects of statins, and they include muscle pain, fatigue and weakness.
Some tests have also shown increased myositis in patients receiving statins (Golomb &
Evans, 2008). Rhabdomyolysis is one of the most serious adverse effects of statins. It
occurs when muscle damage is severe, leading to a marked elevation of creatinine kinase
(CK) (e.g. in excess of 10 times the upper limit of normal). This is often accompanied by
evidence of renal dysfunction and occasionally renal failure and death (Eriksson, Angelin &
Sjberg, 2005).
Mann and Spoerry (1974) observed that Maasai warriors in Africa showed a
reduction in serum cholesterol levels after consumption of large amounts of milk fermented
with a wild Lactobacillus strain. Subsequently, there has been considerable interest in the
beneficial effects of fermented milk products containing lactobacilli and/or bifidobacteria on
human lipid metabolism. Hence, given the research conducted which showed that probiotics
such as Lactobacillus acidophilus are capable of reducing levels of cholesterol (Li, 2012), the

potential of probiotics in lowering the incidence of cardiovascular diseases could be

explored.
Objectives
The objectives of the study are to:
1. Investigate the ability of lactic acid bacteria in removing cholesterol;
2. Investigate the mechanism of cholesterol removal by lactic acid bacteria;
3. Investigate the effectiveness of kidney beans and sunflower seeds in inhibiting HMGCoA reductase in the cholesterol biosynthesis pathway.

Hypotheses
The hypotheses of the study are as follows:
1. Lactic acid bacteria (L. casei, L. acidophilus, L. plantarum and L. lactis) are effective
in removing cholesterol.
2. Cholesterol is removed by lactic acid bacteria via binding to the cell wall.
3. Kidney beans and sunflower seeds are effective in inhibiting HMG-CoA reductase.
Procedure
Apparatus and Materials
Incubator
Shaking incubator
Biological safety cabinet
Autoclave
UV-vis spectrophotometer
Bunsen burner
Mortar and pestle
Microplate reader
Centrifuge
Microcentrifuge
Syringe
0.45 m microfilter
0.85% saline solution

Cholesterol
Tween 20 (10%)
Kidney beans (Phaseolus vulgaris)
Sunflower seeds (Helianthus annuus)
MRS medium
Microtitre plate
Total cholesterol assay kit (Cell Biolabs, Inc)
HMG-CoA reductase assay kit (Sigma-Aldrich)
Lactobacillus casei
Lactobacillus acidophilus
Lactobacillus plantarum
Lactobacillus lactis

Variables

Independent variables
Species of lactic acid

Dependent variables
Absorbance of mixture in

Controlled variables
Concentration and volume of

bacteria

each well at 450 nm

cholesterol

(residual cholesterol assay

and mechanism of
Type of extract

cholesterol removal test)

Absorbance of mixture in

Absorbance of bacteria

each well at 340 nm (HMG-

culture

CoA reductase assay)

Living or non-living bacteria

Concentration and volume of

extract
Temperature of incubation

Growth of bacteria
Lactic acid bacteria were grown in MRS broth at 30C with shaking for 2 days.

Removal of cholesterol by lactic acid bacteria

Cholesterol was dissolved in Tween 20 at a concentration of 10 mg/ml. The resulting
solution was then filter-sterilised. Cholesterol was added to MRS broth at a final
concentration of 0.1 mg/ml. The test set-up contained 9 ml MRS broth with cholesterol and 1
ml of bacterial culture. In the control set-up (without cells), the bacterial culture was replaced
with 1 ml MRS broth.

Five replicates of each set-up were prepared and they were incubated with shaking
at 30C for 3 days. After incubation, 1 ml portions were removed and centrifuged at 12 000

50 l supernatant of
bacteria (grown with
cholesterol)

50 l supernatant of bacteria
with Tween 20 (without

rpm for 10 min to remove the cells. The supernatants were removed and assayed for
residual cholesterol using the Total Cholesterol and Cholesteryl Ester Colorimetric Assay Kit
II (BioVision).

Figure 1: Contents of each well (for test) in residual cholesterol assay

50 L of each sample was added to separate wells of a 96-well micro titer plate. 50

50 l of cholesterol
reaction mix

50 l of cholesterol
reaction mix

Blank (Test)
Test
L cholesterol reaction mix was added to each well and mixed thoroughly. Cholesterol
reaction mix consisted of 46 l cholesterol assay buffer, 2 l substrate mix and 2 l enzyme
mix. For test, 50 l supernatant of lactic acid bacteria (grown with cholesterol) was added to
50 l of cholesterol reaction mix. For the blank prepared for the test setup, 50 l supernatant
of lactic acid bacteria (grown without cholesterol) was added to 50 l of cholesterol reaction
mix. For control, 50 l MRS broth (with cholesterol) was added to 50 l of cholesterol
reaction mix. Finally, for the blank for control, 50 ml MRS broth (without cholesterol) was
added to 50 l of cholesterol reaction mix.

The plate was incubated for 30 min at 37C. It is then read with a spectrophotometric
microplate reader at 450 nm. The amount of residual cholesterol was calculated by
comparing the absorbance values with those of the cholesterol standard curve.

Mechanism of cholesterol removal

Lactic acid bacteria were boiled in a 100oC water bath. The first test setup contains 8
ml of MRS broth, 1 ml of 10 mg/ml cholesterol and 1 ml boiled lactic acid bacteria culture.
For the second test setup, the boiled Lactobacillus culture was replaced with living
Lactobacillus culture. In the control setup (without cells), 9 ml of MRS broth was added to 1
ml of 10 mg/ml cholesterol. Five replicates of each set-up were prepared and they were
incubated with shaking at 30C for 3 days. After centrifugation to pellet the cells, the residual
cholesterol test was performed.

Preparation of extracts
5 g of kidney beans and sunflower seeds were grounded in 15 ml of 0.85% saline solution in
a mortar and pestle. The mixtures were centrifuged at 7000 rpm for 10 min and the
supernatants were collected. The supernatants were filter-sterilised through a 0.45 m
microfilter.

Inhibition of HMG CoA Reductase

The method used is according to that in the HMG- CoA Reductase Assay Kit (SigmaAldrich).
HMG-CoA + 2 NADPH + 2 H+ -> mevalonate + 2 NADP+ + CoA-SH
The following mixtures were prepared in a 96-well microtitre plate:
Volume/l
1x assay buffer

Inhibitor

NADPH

HMG CoA

HMGR

Control

182

12

Blank

184

12

Test

82-132

50-100

12

Blank

84-134

50-100

12

Pravastatin

181

12

Blank

183

12

Five replicates were prepared for each set-up. Contents were mixed well. The absorbance
was taken at 340 nm every 5 min for up to 20 min. The absorbance should decrease due to
a decrease in NADPH concentration if no inhibitor is present. If not, it should remain
constant or decrease at a lower rate than without inhibitor.

Results

Removal of cholesterol by lactic acid bacteria

In the preliminary screening test, the amount of residual cholesterol was higher for control
setups without bacteria as compared to L. casei, L. plantarum and L. lactis. L. plantarum was
the most effective in reducing cholesterol levels, followed by L. lactis and L. casei. This is
shown in Figure 2.

Figure 2: Graph of removal of cholesterol by lactic acid bacteria showed that L. casei, L.
plantarum and L. lactis were effective in reducing cholesterol levels

In another repeat conducted with both L. acidophilus and L. plantarum, the amount of
residual cholesterol was higher for control setups without bacteria as compared to that of
test setups with L. plantarum and L. acidophilus. Once again, L. plantarum was the most
effective in reducing cholesterol levels, followed by L. acidophilus. This is shown in Figure 3.
The Kruskal-Wallis test showed that the amount of residual cholesterol at the end of the
experiment for all three setups were significant, with a p-value of 0.039.

Figure 3: Graph of removal of cholesterol by lactic acid bacteria showed that L. casei and L.
acidophilus were effective in reducing cholesterol levels to a different extent.

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Mechanism of cholesterol removal

For the mechanism of cholesterol removal test, there was a lower amount of residual
cholesterol in control setups without bacteria, as compared to that in the presence of living
L. plantarum and non-living L. plantarum.
The Kruskal-Wallis test showed that there was a significant difference in the amount of
residual cholesterol formed in the three groups, with a p-value of 0.004. However, it should
be noted that non-living L. plantarum was not as effective as living L. plantarum in reducing
cholesterol levels. This is shown in Figure 4.

Figure 4: The mechanism of cholesterol removal test showed that living L. plantarum was
more effective than non-living L. plantarum in reducing cholesterol levels.

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Inhibition of HMG CoA Reductase

For the inhibition of HMG-CoA reductase experiment, the activity of the enzyme HMG-CoA
reductase was determined. This is shown in Figure 5. From the graph, sunflower seed
extract showed great effectiveness in inhibiting HMG-CoA reductase. The Kruskal-Wallis test
showed that the difference in HMG-CoA reductase activity amongst the three setups was
significant, with a p-value of 0.003.

Figure 5: The inhibition of HMG-CoA reductase test showed that sunflower seed extract was
strongly inhibitory towards HMG-CoA reductase.

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In a repeat conducted with varying amounts of kidney bean extract, HMG-CoA reductase
was inhibited to a greater extent in the presence of 0.10 ml kidney bean extract, as
compared to that of 0.05 ml kidney bean extract and control without inhibitor. This is shown
in Figure 6.

Figure 6: The inhibition of HMG-CoA reductase test showed that kidney extract was
inhibitory towards HMG-CoA reductase.

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In a final repeat conducted with 80 l of kidney bean extract, HMG-CoA reductase activity
was higher for control without inhibitor as compared to that of kidney bean extract and
pravastatin. This is shown in Figure 7. The difference in HMG CoA reductase activity was
significant, as shown by the Kruskal-Wallis test, with a p-value of 0.012.

Figure 7: The inhibition of HMG-CoA reductase test showed that kidney extract was
inhibitory towards HMG CoA reductase.

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Conclusion
From the study, it can be seen that L. plantarum are more effective than L. lactis,
L. acidophilus and L. casei in reducing cholesterol levels. Furthermore, L. plantarum
removes cholesterol by allowing cholesterol to bind onto its cell wall and assimilation by L.
plantarum cells, based on results showing that both living and non-living cells exhibited
cholesterol reducing properties and that living cells removed more cholesterol than non-living
cells, as non-living cells could not assimilate cholesterol, but were able to allow it to bind to
its cell wall. Kidney beans and sunflower seeds have also been shown to be effective in
reducing cholesterol levels via the inhibition of the enzyme HMG-CoA reductase. Hence the
consumption of increased amounts of lactic acid bacteria, kidney beans and sunflower seeds
to overcome the problem of high cholesterol levels in humans shows great potential.

This study utilises natural substances such as lactic acid bacteria, sunflower seeds
and kidney beans to remove cholesterol, the intake of which can be increased by a change
in diet, hence is virtually zero cost, commonly available and with few known side effects.
This also reduces the usage of statins, which causes muscle pain, fatigue and weakness.
(Golomb & Evans, 2008) Our results were comparable to that of Guo, Yang and Huo (2011),
who showed that L. plantarum was effective in removing cholesterol. The results for the
mechanism of cholesterol removal in lactic acid bacteria also complemented the study
conducted by Li (2012), who showed that probiotics such as Lactobacillus acidophilus were
capable of reducing levels of cholesterol via coprecipitation and assimilation, as well as the
adsorption and incorporation of cholesterol to membrane. Li also recognised two other
cholesterol removing mechanisms of probiotics, namely reducing the host absorption of
cholesterol and the deconjugation of bile salts.

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One limitation of the study is the inability to conduct the tests in vivo, due to
complications with experimentation on humans, hence the reproducibility of results in
practice cannot be determined.
This study has potential applications such as recommending a change in diet for
patients facing high cholesterol problems to include more kidney beans and sunflower
seeds, such that the body produces less cholesterol, reducing cholesterol levels. We can
also encourage the intake of lactic acid bacteria with cholesterol-rich meals to remove
cholesterol before absorption, such as through cultured milk, hence allowing less cholesterol
to be absorbed into the bloodstream, lowering cholesterol levels. This reduces the use of
statins which poses adverse effects to human health such as causing pain, fatigue and
rhabdomyolysis. It also provides a cheaper alternative for patients with high blood
cholesterol as a change in diet is low cost compared to purchasing commercial drugs.

Some future work for consideration include investigating the effect of bile salts on
cholesterol removal as it is one of the methods described by Pereira and Gibson (2002), and
is present naturally in the alimentary canal, to further analyse the mechanism of removal of
cholesterol by lactic acid bacteria. We can also repeat the experiment with other species of
lactic acid bacteria to see which species is the most effective in terms of cholesterol removal,
allowing patients to choose from the most effective species of bacteria and encouraging
producers of cultured milk to incorporate such bacteria into their products.

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References

[1]

Eriksson M., Angelin B. and Sjberg S. (2005). Risk for fatal statin-induced
rhabdomyolysis as a consequence of misinterpretation of 'evidence-based medicine'.
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http://www.ncbi.nlm.nih.gov/pubmed/15715689/

[2]

Golomb, B.A. and Evans, M.A. (2008). Statin Adverse Effects: A Review of the
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[3]

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[4]

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[6]

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[8]

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Acknowledgements

We are grateful to Hwa Chong Institution for providing the facilities to carry out this work; our
mentor, Mrs Goh-Yip Cheng Wai, for her valuable guidance throughout this project; Mdm Lim
Cheng Fui and Mr Xie Shun Quan of the SRC Biology lab for their assistance while
conducting experiments.

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