In this study, we used everyday food items such as lactic acid bacteria (found in yogurt), sunflower seeds and kidney beans, to either absorb cholesterol in the gut or inhibit cholesterol synthesis in the body. Findings can potentially provide doctors and health experts with an alternative to statins, cholesterol-lowering drugs that may lead to side effects. As future work, we can explore the chemical compound present in the natural extracts that help inhibit cholesterol synthesis, and if possible, carry out trials in vivo to better determine the effects of these extracts.

1. Ng De Rong Tony, Chay Hui Xiang, Lai Tian Lang
Background of Research Area
According to 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 HMG-
CoA reductase, can effectively reduce cholesterol levels and treat patients with high
cholesterol, they are known to have severe side effects, such as muscle pain, fatigue and
weakness (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 & Sjöberg, 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.
Purpose of Research Area
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 HMG-
CoA reductase in the cholesterol biosynthesis pathway.

2. Ng De Rong Tony, Chay Hui Xiang, Lai Tian Lang
50 ul supernatant of
bacteria (grown with
cholesterol)
50 ul of cholesterol
reaction mix
50 ul of cholesterol
reaction mix
50 ul supernatant of
bacteria with Tween 20
(without cholesterol)
Test Blank
Figure 1: Contents of each well (for test) in residual cholesterol assay
Hypotheses of Research
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.
Methods
Growth of bacteria
Lactic acid bacteria were grown in MRS broth at 30°C 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 setup contained 9 ml MRS broth with cholesterol and 1
ml of bacterial culture. In the control setup (without cells), the bacterial culture was replaced
with MRS broth.
Five replicates of each setup were prepared and they were incubated with shaking at
30°C for 3 days. After incubation, 1 ml portions were centrifuged at 12 000 rpm for 10 min to
remove the cells. The supernatants were assayed for residual cholesterol using the Total
Cholesterol and Cholesteryl Ester Colorimetric Assay Kit II (BioVision).
50 µL of each sample was added to separate wells of a 96-well micro titer plate. 50
µL cholesterol reaction mix was added to each well and mixed thoroughly. Cholesterol
reaction mix consisted of 46 µl cholesterol assay buffer, 2 µl substrate mix and 2 µl enzyme
mix. For test, 50 µl supernatant of lactic acid bacteria (grown with cholesterol) was added to
50 µl of cholesterol reaction mix. For the blank prepared for the test setup, 50 µl supernatant
of lactic acid bacteria (grown without cholesterol) was added to 50 µl of cholesterol reaction
mix. For control, 50 µl MRS broth (with cholesterol) was added to 50 µl of cholesterol
reaction mix. Finally, for the blank for control, 50 ml MRS broth (without cholesterol) was
added to 50 µl of cholesterol reaction mix.

3. Ng De Rong Tony, Chay Hui Xiang, Lai Tian Lang
HMG-CoA + 2 NADPH + 2 H+ -> mevalonate + 2 NADP+
+ CoA-SH
The plate was incubated for 30 min at 37ºC. It was 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 contained 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 setup were prepared and they were
incubated with shaking at 30°C 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 in a mortar
and pestle. The mixtures were centrifuged at 7000 rpm for 10 min and the supernatants were
collected, which were filter-sterilised through a 0.45 μm microfilter.
Inhibition of HMG CoA Reductase
The method used was according to that in the HMG- CoA Reductase Assay Kit (Sigma-
Aldrich).
The following mixtures were prepared in a 96-well microtitre plate:
Five replicates were prepared for each setup. Contents were mixed well. The absorbance was
taken at 340 nm every 5 min for up to 20 min.

4. Ng De Rong Tony, Chay Hui Xiang, Lai Tian Lang
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
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.
Figure 4: The mechanism of cholesterol removal test
showed that living L. plantarumwas more effective than
non-living L. plantarumin reducing cholesterol levels.
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. (See Figure 2).
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.
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.

5. Ng De Rong Tony, Chay Hui Xiang, Lai Tian Lang
Figure 6: The inhibition of HMG-CoA reductasetest showed that
kidney extract was inhibitory towards HMG-CoA reductase.
Figure 5: The inhibition of HMG-CoA reductasetest showed that
sunflower seed extract was strongly inhibitory towards HMG-CoA
reductase.
Figure 7: The inhibition of HMG-CoA reductase test
showed that kidney extract was inhibitory towards
HMG CoA reductase.
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.
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.
Lastly, a repeat conducted with 80 μl of kidney bean extract showed that 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.

6. Ng De Rong Tony, Chay Hui Xiang, Lai Tian Lang
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
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 shown to be effective in reducing
cholesterol levels via the inhibition of the enzyme. 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
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), which
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 cholesterol levels 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: reducing the host absorption of cholesterol
and the deconjugation of bile salts.
One limitation of the study is inability to conduct the tests in vivo, due to
complications with experimentation on humans, hence compromising the reproducibility of
results.
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 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,
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

7. Ng De Rong Tony, Chay Hui Xiang, Lai Tian Lang
alternative for patients with high 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 repeat the experiment with other species of lactic
acid bacteria to see which species is the most effective, allowing patients to choose from the
most effective species of bacteria and encouraging producers to utilise such bacteria for their
products.
References
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http://www.ncbi.nlm.nih.gov/pubmed/15715689/
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8. Ng De Rong Tony, Chay Hui Xiang, Lai Tian Lang
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