Cholesterol-Lowering Probiotics as Potential Biotherapeutics for Metabolic Diseases

Abstract

Cardiovascular diseases are one of the major causes of deaths in adults in the western world. Elevated levels of certain blood lipids have been reported to be the principal cause of cardiovascular disease and other disabilities in developed countries. Several animal and clinical trials have shown a positive association between cholesterol levels and the risks of coronary heart disease. Current dietary strategies for the prevention of cardiovascular disease advocate adherence to low-fat/low-saturated-fat diets. Although there is no doubt that, in experimental conditions, low-fat diets offer an effective means of reducing blood cholesterol concentrations on a population basis, these appear to be less effective, largely due to poor compliance, attributed to low palatability and acceptability of these diets to the consumers. Due to the low consumer compliance, attempts have been made to identify other dietary components that can reduce blood cholesterol levels. Supplementation of diet with fermented dairy products or lactic acid bacteria containing dairy products has shown the potential to reduce serum cholesterol levels. Various approaches have been used to alleviate this issue, including the use of probiotics, especially Bifidobacterium spp. and Lactobacillus spp.. Probiotics, the living microorganisms that confer health benefits on the host when administered in adequate amounts, have received much attention on their proclaimed health benefits which include improvement in lactose intolerance, increase in natural resistance to infectious disease in gastrointestinal tract, suppression of cancer, antidiabetic, reduction in serum cholesterol level, and improved digestion. In addition, there are numerous reports on cholesterol removal ability of probiotics and their hypocholesterolemic effects. Several possible mechanisms for cholesterol removal by probiotics are assimilation of cholesterol by growing cells, binding of cholesterol to cellular surface, incorporation of cholesterol into the cellular membrane, deconjugation of bile via bile salt hydrolase, coprecipitation of cholesterol with deconjugated bile, binding action of bile by fibre, and production of short-chain fatty acids by oligosaccharides. The present paper reviews the mechanisms of action of anti-cholesterolemic potential of probiotic microorganisms and probiotic food products, with the aim of lowering the risks of cardiovascular and coronary heart diseases.

Full text

Hindawi Publishing Corporation
Experimental Diabetes Research
Volume 2012, Article ID 902917, 14 pages
doi:10.1155/2012/902917
Review Article
Cholesterol-Lowering Probiotics as Potential Biotherapeutics
for Metabolic Diseases
Manoj Kumar,1Ravinder Nagpal,2Rajesh Kumar,1R. Hemalatha,1
Vinod Verma,3Ashok Kumar,4Chaitali Chakraborty,5Birbal Singh,6
Francesco Marotta,7Shalini Jain,8and Hariom Yadav9
1Department of Microbiology & Immunology, National Institute of Nutrition, Hyderabad 50007, India
2Shaheed Udham Singh College of Research & Technology, Punjab, Mohali, Radaur, Haryana, India
3Research and Development Unit, National Heart Centre, Singapore 1687521
4Department of Zoology, M.L.K. Post-Graduate College, Balrampur 271201, India
5Department of Biotechnology, ITS Paramedical College, Ghaziabad 201206, India
6Indian Veterinary Research Institute, Regional Station, Palampur 176061, India
7Hepato-Gastroenterology Unit, S. Giuseppe Hospital, Vittore, 20123 Milano, Italy
8Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, MD 20892, USA
9Endocrinology, Diabetes, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, MD 20892, USA
Correspondence should be addressed to Shalini Jain, shalini2601@gmail.com and Hariom Yadav, yadavhariom@gmail.com
Received 18 October 2011; Accepted 10 January 2012
Academic Editor: Raffaele Marfella
Copyright © 2012 Manoj Kumar et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Cardiovascular diseases are one of the major causes of deaths in adults in the western world. Elevated levels of certain blood
lipids have been reported to be the principal cause of cardiovascular disease and other disabilities in developed countries. Several
animal and clinical trials have shown a positive association between cholesterol levels and the risks of coronary heart disease.
Current dietary strategies for the prevention of cardiovascular disease advocate adherence to low-fat/low-saturated-fat diets.
Although there is no doubt that, in experimental conditions, low-fat diets offer an effective means of reducing blood cholesterol
concentrations on a population basis, these appear to be less effective, largely due to poor compliance, attributed to low palatability
and acceptability of these diets to the consumers. Due to the low consumer compliance, attempts have been made to identify
other dietary components that can reduce blood cholesterol levels. Supplementation of diet with fermented dairy products or
lactic acid bacteria containing dairy products has shown the potential to reduce serum cholesterol levels. Various approaches have
been used to alleviate this issue, including the use of probiotics, especially Bifidobacterium spp. and Lactobacillus spp.. Probiotics,
the living microorganisms that confer health benefits on the host when administered in adequate amounts, have received much
attention on their proclaimed health benefits which include improvement in lactose intolerance, increase in natural resistance to
infectious disease in gastrointestinal tract, suppression of cancer, antidiabetic, reduction in serum cholesterol level, and improved
digestion. In addition, there are numerous reports on cholesterol removal ability of probiotics and their hypocholesterolemic
effects. Several possible mechanisms for cholesterol removal by probiotics are assimilation of cholesterol by growing cells, binding
of cholesterol to cellular surface, incorporation of cholesterol into the cellular membrane, deconjugation of bile via bile salt
hydrolase, coprecipitation of cholesterol with deconjugated bile, binding action of bile by fibre, and production of short-chain
fatty acids by oligosaccharides. The present paper reviews the mechanisms of action of anti-cholesterolemic potential of probiotic
microorganisms and probiotic food products, with the aim of lowering the risks of cardiovascular and coronary heart diseases.
1. Introduction
Although cholesterol is an important basic block for body
tissues, elevated blood cholesterol is a well-known major
risk factor for coronary heart diseases [1]. WHO has pre-
dicted that, by 2030, cardiovascular diseases will remain the
leading causes of death, affecting approximately 23.6 million
people around the world [2]. It has been reported that

2Experimental Diabetes Research
hypercholesterolemia contributes to 45% of heart attacks
in Western Europe and 35% of heart attacks in Central
and Eastern Europe [3]. The risk of heart attack is three
times higher in those with hypercholesterolemia, compared
to those who have normal blood lipid profiles. The WHO
delineated that unhealthy diets, such as those high in fat,
salt, and free sugar and low in complex carbohydrates,
fruits, and vegetables, lead to increased risk of cardiovascular
diseases [4]. Recent modalities for lowering blood cholesterol
levels involve dietary management, behavior modification,
regular exercise, and drug therapy [5]. Pharmacological
agents that effectively reduce cholesterol levels are available
for the treatment of high cholesterol; however, they are
expensive and are known to have severe side effects [6].
Lactic acid bacteria (LAB) with active bile salt hydrolase
(BSH) or products containing them have been suggested to
lower cholesterol levels through interaction with host bile
salt metabolism [7]. Lactobacilli with BSH activity have an
advantage to survive and colonize the lower small intestine
where the enterohepatic cycle takes place, and therefore BSH
activity may be considered as an important colonization
factor [8]. Sanders [9] proposed the mechanism based on
the ability of certain probiotic lactobacilli and bifidobacteria
to deconjugate bile acids enzymatically, increasing their rates
of excretion. Cholesterol, being a precursor of bile acids,
converts its molecules to bile acids replacing those lost
during excretion leading to a reduction in serum cholesterol.
This mechanism could be operated in the control of serum
cholesterol levels by conversion of deconjugated bile acids
into secondary bile acids by colonic microbes. The use of
such orally applied microorganisms (probiotics) is a major
aim of the concept of functional food [10,11]. Recently, there
has been much interest in LAB, especially lactobacilli, due
to their beneficial effects in health including anti-cholesterol,
antidiabetic, antipathogenic, and anticarcinogenic properties
and stimulation of the immune system [10,12–19]. Lacto-
bacillus plantarum, the predominating Lactobacillus species
on oral and intestinal human mucosa, has shown the ability
to survive the passage through the human gastrointestinal
tract and to establish itself for at least a shorter time in the
intestine after consumption [12,16,20].
Lactobacilli are frequently used in products for human
consumption and can be found as probiotics in infant foods,
cultured milks, and various pharmaceutical preparations
[10,21,22]. One beneficial effect that has been suggested
to result from human consumption of probiotic LAB is a
reduction in serum cholesterol levels, as suggested by the
results of several human and animal studies [23]. This effect
can partially be ascribed to an enzymatic deconjugation of
bile acids [24–27]. Deconjugated bile salts are less soluble
and less efficiently reabsorbed from the intestinal lumen than
their conjugated counterparts, which results in excretion of
larger amounts of free bile acids in feces [28,29]. Also, free
bile salts are less efficient in the solubilization and absorption
of lipids in the gut [30]. Therefore, the deconjugation of
bile acids by LAB bacteria could lead towards a reduction
in serum cholesterol either by increasing the demand of
cholesterol for de novo synthesis of bile acids to replace
that lost in feces or by reducing cholesterol solubility and,
Tab le 1: List of some potential bacteria showing bile salt hydrolase
(BSH) activity.
Probiotic organisms with BSH activity References
Bifidobacterium adolescentis [38]
B. animalis [38]
B. breve [38]
B. infantis [39]
B. longum [39]
Bifidobacterium sp. [40,41]
Lactobacillus acidophilus [41–43]
L. casei [41–43]
L. fermentum
L. gasseri [38,41]
L. helveticus [38]
L. paracasei subsp. paracasei [44]
L. rhamnosus [38,44]
L. plantarum [8,19]
thereby, absorption of cholesterol throughout the intestinal
lumen. Moreover, Gilliland et al. [31] observed a significant
relationship between cholesterol assimilation by probiotic
lactobacilli and their degree of bile deconjugation. BSH,
the enzyme responsible for bile salt deconjugation during
enterohepatic circulation, has been detected in several LAB
species indigenous to the gastrointestinal tract (Tabl e 1 )[7,
28,32,33]. It has also been suggested that BSH activity
should be a requirement in the selection of probiotic
organisms with cholesterol-lowering properties, as non-
deconjugating organisms do not appear to be able to remove
cholesterol from the culture medium to any significant extent
[26]. Lactobacillus fermentum, a normal resident of the
human gut microflora, has been reported to adhere to the
epithelial cells, with a preference for the small intestine [34].
It has also been shown to colonize the intestine after oral
administration [35] and produce surface-active components
that inhibit the adhesion of uropathogenic bacteria [36,37]
(Tabl e 2).
2. Bile
Bile is a yellow-green aqueous solution whose major con-
stituents include bile acids, cholesterol, phospholipids, and
the pigment biliverdin [58,59]. It is synthesized in the
pericentral hepatocytes of the liver, stored and concentrated
in the gallbladder interdigestively, and released into the duo-
denum after food intake. Bile functions as a biological deter-
gent that emulsifies and solubilizes lipids, thereby playing an
essential role in fat digestion. This detergent property of bile
also confers potent antimicrobial activity, primarily through
the dissolution of bacterial membranes [60,61]. Bile acids
are saturated, hydroxylated C-24 cyclopentanophenanthrene
sterols synthesized from cholesterol in hepatocytes. The two
primary bile acids synthesized in the human liver are cholic
acid (CA; 3a,7a,12a-trihydroxy-5b-cholan-24-oic acid) and

Experimental Diabetes Research 3
Tab le 2: Summary of major findings for probiotic mediated cholesterol reduction.
S. No. Probiotic organism Experimental system Major findings Reference
1 Unknown (fermented milk) Maasai tribesmen in Africa Low cholesterol [45]
2 Unknown (Yogurt) Human subjects Reduced cholesterol [46]
3Lactobacillus acidophilus Culture media
Cholesterol removal
Better survival in cholesterol
media
[31]
4Bifidobacterium Culture media Removal of cholesterol [25]
5L. acidophilus Culture media Cholesterol assimilation [47]
6 Probiotic fermented milk Rats Cholesterol reducing efficacy [48]
7L. reuteri Mice Reduced blood cholesterol
Decreased triglycerides [49]
8Bifidobacterium milk Rats, Human
Reduced cholesterol
Decreased triglyceride
Decreased LDL
Increased HDL
[50]
9Yoghurt containing B. lactis or
B. longum Rats
Reduced cholesterol
Decreased triglyceride
Decreased LDL
Increased HDL
[51]
10 L. plantarum Culture media Cholesterol assimilation [52]
11 L. bulgaricus and L. acidophilus Human Decreased cholesterol Lin et al. [53]
12 Lactobacillus sporogenes Human Decreased Cholesterol
Reduced LDL-cholesterol [54].
13 L. acidophilus Human Decreased cholesterol Gilliland [55]
14 E. faecium Human
Decreased cholesterol levels
Decreased triglyceride
Decreased LDL
Increased HDL
[56]
15
Microencapsulated bile salt
hydrolase- (BSH-) active
Lactobacillus reuteri NCIMB 30242
Human
Reduced LDL-cholesterol
Decreased total
cholesterol
Decreased apoB-100
Decreased
non-HDL-cholesterol
[57]
chenodeoxycholic acid (CDCA; 3a,7a-dihydroxy-5b-cholan-
24-oic acid). Bile acids are further metabolized by the liver
via conjugation (N-acyl amidation) to glycine or taurine,
a modification that decreases the Pka to approximately 5.
Thus, at physiological pH, conjugated bile acids are almost
fully ionized and may be termed bile salts [62]. The primary
bile acids, cholic and chenodeoxycholic acid, are synthesized
de novo in the liver from cholesterol. The solubility of the
hydrophobic steroid nucleus is increased by conjugation as
an N-acyl amidate with either glycine (glycoconjugated) or
taurine (tauroconjugated) prior to secretion. The resulting
molecules are therefore amphipathic and can solubilize
lipids to form mixed micelles. Bile acids are efficiently
conserved under normal conditions by a process termed
enterohepatic recirculation. Conjugated and unconjugated
bile acids are absorbed by passive diffusion along the entire
gut and by active transport in the terminal ileum [58].
Reabsorbed bile acids enter the portal bloodstream and
are taken up by hepatocytes, reconjugated, and resecreted
into bile. Approximately 5% of the total bile acid pool
(0.3 to 0.6 g) per day eludes epithelial absorption and may
be extensively modified by the indigenous intestinal bacteria
[63]. One important transformation is deconjugation, a
reaction that must occur before further modifications are
possible [64]. Deconjugation is catalyzed by BSH enzymes
(EC 3.5.1.24), which hydrolyze the amide bond and liberate
the glycine/taurine moiety from the steroid core (Figure 1).
The resulting acids are termed unconjugated or deconjugated
bile acids.
2.1. Identification of bsh Homologs in Probiotic Genomes.
The genes that may encode BSH enzymes in the genome
sequences of potential probiotic bacteria are available
in public databases (National Center for Biotechnology
Information genome site (http://www.ncbi.nlm.nih.gov/)
and the Joint Genome Institute microbial genomics site
(http://genome.jgi-psf.org/)). Several strains (e.g., Lacto-
bacillus plantarum WCFS1) possess more than one BSH
homolog, which are not identical. The genetic geography of
bsh regions is not the same in all strains, and in cases where

4Experimental Diabetes Research
Precursor of bile salts
(cholesterol)
De novo synthesis
Taurine conjugated bile salts
Glycine conjugated bile salts
Systemic circulation
Bile duct
Hydrolysis by bsh
Free cholic acid
Amino acid group
Reabsorbed into intestine
Excretion in feces
Dietary fats
Glycine
Taurine
GI tract
Cholesterol
absorption
Probiotic bacteria cleave
conjugates or adsorb cholesterol
on surface
Figure 1: Cholesterol as the precursor for the synthesis of new bile acids and the hypocholesterolemic role of bile salt hydrolase (BSH).
more than one is present they are not located in the same
region of the chromosome.
2.2. bsh Genes in Probiotic Bacteria. Since variability in bsh
phenotypes has been observed within isolates of some species
[38,42,65,66], it has been speculated that bsh genes may
have been acquired horizontally [65]. Comparison of the
bsh gene and surrounding sequences of L. acidophilus strain
KS-13 and L. johnsonii 100-100 by Elkins et al. [65]has
revealed little or no synteny flanking this locus. It was also
noted that L. johnsonii 100-100 encodes a group II intron
protein (maturase mat)downstreamofbsh. In addition to
reverse transcriptase activity, these proteins can function
as maturases and endonucleases and facilitate movement
and splicing of cDNA into the genome. Group II intron
proteins are often inserted in or associated with mobile
genetic elements [67]. Sequencing of the entire genome of
L. acidophilus NCFM has revealed that this strain possesses
two bsh genes (bshA and bshB). The predicted sequence
of the BSH enzymes encoded by these loci share a higher
level of similarity to BSH enzymes from other Lactobacillus
species than to each other, suggesting that they may have
been acquired from different sources [68]. In short, BSH is
present in all bifidobacterial strains and lactobacilli strains
associated with the gastrointestinal environment, but bsh
genes can potentially be acquired from these strains by other
intestinal microorganisms (e.g., L. monocytogenes).
3. Functions of BSH
The precise function(s) of microbial BSHs is currently not
understood, although several hypotheses have been pro-
posed, as follows.
3.1. Nutritional Role. The amino acids liberated from bile
salt deconjugation could potentially be used as carbon,
nitrogen, and energy sources, since glycine may be metab-
olized to ammonia and carbon dioxide, and taurine may
be metabolized to ammonia, carbon dioxide, and sulfate.
Bile salt deconjugation may therefore confer a nutritional
advantage on hydrolytic strains. In support of this hypoth-
esis, Huijghebaert et al. [69] and Van Eldere et al. [70]
observed that certain BSH-positive strains of Clostridium
utilized the released taurine as an electron acceptor, and
growth rates were improved in the presence of taurine
and taurine-conjugated bile salts. It has also been noted
that transcription of the Bifidobacterium longum bsh gene
is coupled to a homolog of glnE that encodes a glutamine
synthetase adenyltransferase that forms part of the nitrogen
regulation cascade [39]. However, experiments performed by
Tannock et al. [71] and Gilliland and Speck [72] refute this
hypothesis since these authors observed that the lactobacilli
used in their studies did not utilize the steroid moiety of the
bile salt for cellular precursors since neither ring cleavage nor
subsequent metabolism occurred.
3.1.1. Alteration of Membrane Characteristic. The bacteri-
olytic enzymes lysozyme and phospholipase A2, and an-
timicrobial peptides such as α-defensins, are important
contributors to innate immunity in the intestine. The
composition, fluidity, permeability, hydrophobicity, and net
charge of bacterial membranes all determine the extent
of damage by these host defenses. It has been proposed
that BSHs facilitate incorporation of cholesterol or bile
into bacterial membranes [73–75]. This incorporation may
increase the tensile strength of the membranes [76]ormay
change their fluidity or charge. Cell surface modifications
that may result from BSH activity could potentially offer

Experimental Diabetes Research 5
protection against perturbation of the structure and integrity
of bacterial membranes by the immune system, and such
resistance mechanisms may be important in establishing
persistent infections. Such a function may strongly select
for commensals possessing BSH enzymes while mitigating
against BSH-negative pathogens or other transients.
3.1.2. Bile Detoxification. Studies by various research groups
using wild-type and bsh mutant pairs have provided a link
between bile salt hydrolysis and bile tolerance. A Lactobacillus
amylovorus mutant with a partial decrease in BSH activity
isolated using an N-methyl-N1-nitro-N-nitrosoguanidine
mutagenesis strategy displayed decreased growth rates in
the presence of bile salts [40]. Also, mutation of bsh in
Lactobacillus plantarum [8]andListeria monocytogenes [60,
61] renders cells significantly more sensitive to bile and bile
salts. The precise mechanism by which BSH enzymes play
a role in the tolerance of bile is not yet fully understood.
However, it has been proposed that since the protonated
(non-dissociated) form of bile salts may exhibit toxicity
through intracellular acidification in a manner similar to
organic acids, BSH-positive cells may protect themselves
through the formation of the weaker unconjugated coun-
terparts [77]. This could help negate the drop in pH
by recapturing and exporting the cotransported proton.
The ratio of glycoconjugated to tauroconjugated bile salts
in human bile is usually 3 : 1. In vitro experiments have
revealed that whereas tauroconjugated bile salts usually only
have slight affects(ifany)onbacterialcellsateverypH
examined, glycoconjugated bile salts are extremely toxic at
acidic pHs, and bsh mutants are significantly more inhibited
than corresponding parent cells [60,61,77]. Therefore, it
has been suggested that BSHs are particularly important in
combating the toxic effects of glycoconjugated bile salts at
low pH, and BSH activity may be of particular importance
at the point where bile enters the duodenum and where
acid reflux may occur from the stomach or in localized
microenvironments in the intestine when the pH is lowered
by lactic acid bacteria. The fact that BSHs have been shown
to preferentially hydrolyze glycoconjugated bile salts [78,79],
together with the observation that BSHs have slightly acidic
pH optima (usually between pH 5 and 6) [42,80], may serve
to substantiate this theory.
3.1.3. Gastrointestinal Persistence. Since BSHs may combat
the deleterious effects of bile (and perhaps components of
the innate immune system such as the defensins through
cell surface modifications), a role for these enzymes in
survival/persistence of strains within the gastrointestinal
tract is conceivable. Bateup et al. [81] compared the abilities
of three Lactobacillus strains which demonstrated various
degrees of BSH activity in vitro (one strain demonstrated
high activity, one showed moderate activity, and one lacked
activity) to colonize Lactobacillus-free mice. Enumeration
of lactobacilli in the gastrointestinal organs 2 weeks after
inoculation revealed that all strains colonized equally well,
leading to the conclusion that BSH is not essential for
colonization. However, a more recent study by Dussurget et
al. [82] convincingly demonstrates that BSH contributes to
persistence of L. monocytogenes within the gastrointestinal
tract. A bsh mutant demonstrated reduced bacterial fecal
carriage after oral infection of guinea pigs (counts of the
mutant were 4 to 5 logs lower than the parent after
48 h). It was also observed that intestinal multiplication
of the parent could be increased approximately 10-fold
by supplying cells with an extra copy of the gene on a
plasmid, further confirming the importance of BSH to
intestinal persistence [82]. Two obvious differences between
this L. monocytogenes study and the earlier one of Bateup
et al. [81] may account for their different conclusions.
First, isogenic L. monocytogenes wild-type and bsh mutant
strains were compared, and it is possible that intrinsic
differences between the strains of lactobacilli used in the
other study masked the contribution of BSH to intestinal
survival. Furthermore, Bateup et al. [81] used Lactobacillus-
free mice, and it is possible that a role for BSH would be
uncovered in a more competitive environment. Therefore,
future investigations with bifidobacterial and Lactobacillus
bsh mutants would be necessary to unequivocally determine
whether gastrointestinal persistence is a universal function of
BSHs.
3.2. Impact of Microbial BSH Activity on the Host
3.2.1. Cholesterol Lowering. Hypercholesterolemia (elevated
blood cholesterol levels) is considered a major risk factor
for the development of coronary heart disease, and although
pharmacologic agents are available to treat this condition
(e.g., statins or bile acid sequestrants), they are often
suboptimal and expensive and can have unwanted side effects
[83]. Oral administration of probiotics has been shown
tosignificantlyreducecholesterollevelsbyasmuchas22
to 33% [7,23] or prevent elevated cholesterol levels in
mice fed a fat-enriched diet [84]. These cholesterol-lowering
effects can be partially ascribed to BSH activity (other
possible mechanisms include assimilation of cholesterol by
the bacteria, binding of cholesterol to the bacterial cell
walls, or physiological actions of the end products of short-
chain fatty acid fermentation (Figure 2)) [80]. Deconjugated
bile salts are less efficiently reabsorbed than their conju-
gated counterparts, which results in the excretion of larger
amounts of free bile acids in feces. Also, free bile salts are
less efficient in the solubilization and absorption of lipids
in the gut. Therefore, deconjugation of bile salts could lead
to a reduction in serum cholesterol either by increasing the
demand for cholesterol for de novo synthesis of bile acids to
replace those lost in feces or by reducing cholesterol solubility
and thereby absorption of cholesterol through the intestinal
lumen.
Impaired Digestive Functions. Since unconjugated bile acids
are less efficient than conjugated molecules in the emul-
sification of dietary lipids and the formation of micelles,
BSH activity may compromise normal lipid digestion and
the absorption of fatty acids and monoglycerides could be

6Experimental Diabetes Research
SCFA
Probiotics
Prebiotics
Fermentation
Reduced pH
Increased mineral absorption
Fewer toxic bacterial
metabolites
Reduced cancer risk
Colonocytes
Antagonism of
pathogens and
putrefactive bacteria
Epigenetic regulation
HDAC
inhibitor
Controlled serum lipids and
cholesterol
De novo lipogenesis
Reduced colon cancer ris
k
and IBD inflammation
Ca++Mg++
+Trophic and anti-neoplastic
effect
Figure 2: Role of probiotics’ metabolites as epigenetic approach to control high cholesterol and colon cancer.
impaired [28]. Microbial BSH activity has been related to
growth defects in chickens [85] but not in mice [81].
4. Effects of Probiotics on Plasma Lipids
The idea about the health advantages of fermented milk
products in humans goes back to the early 19th century,
when it was proposed by Metchnikov that fermenting milks
by lactic acid bacteria “prevented intestinal putrefaction” and
“helped maintain the forces of the body” [22,86]. A study on
Maasai tribesmen in Africa who have low serum cholesterol
showed that they rarely experience coronary heart diseases,
despite eating a great deal of meat. They regularly consumed
4-5 liters of fermented whole milk per day. This provided
the motivation for investigating fermented milk’s possible
influence on blood cholesterol [45]. Later, in a study by
Mann [46], on twenty-six volunteers, it was found that large
amount of yoghurt reduced cholesterolemia, which could
be due to a factor in yoghurt that prevents production of
cholesterol from acetate. This factor may be either orotic acid
or 3-hydroxy-3-methylglutaric acid plus thermophilus milk
or methanol soluble of thermophilus milk. Gilliland et al.
[31] showed that some strains of Lactobacillus acidophilus
make it possible for cholesterol to be bound to intestine’s
lumen and as a result decrease its absorption. Tahri et al. [25]
investigated assimilation of cholesterol by Bifidobacterium
strains and observed that the removal of cholesterol from
the growth medium is caused by both bacterial activity and
precipitation of cholesterol. Lin and Chen [47]investigated
cholesterol-reducing abilities of L. acidophilus and found that
hypocholesterolemic ability is because of the assimilation of
cholesterol by L. acidophilus cells or its attachment to the
surface of L. acidophilus cells. Grunewald [48]observeda
strong reduction in serum cholesterol of probiotic fermented
milk-fed rats, indicating that cholesterol level in serum can
be reduced by consumption of probiotics. A different study
conducted on mice with high cholesterol demonstrated that
L. reuteri wasabletoreducebloodtriglycerideby38%and
cholesterol by 40% and raised the HDL/LDL cholesterol ratio
by 20% [49]. Xiao et al. [50] also observed that consumption
of Bifidobacterium milk leads to a meaningful reduction in
triglyceride, low-density lipid, and total cholesterol. Similar
results were observed by Abd El-Gawad et al. [51]ina
study on rats fed with yoghurt containing B. lactis or B.
longum. Recently, Kumar et al. [52] also explored probiotic
L. plantarum with potential to control hypercholesterolemia.
Lin et al. [53] conducted human studies and observed that
blood cholesterol was reduced significantly in volunteers

Experimental Diabetes Research 7
who were given tablets of L. bulgaricus and L. acidophilus
for 16 weeks every day. In another study, hyperlipidemic
patients that were given Lactobacillus sporogenes for 90
days showed a 35% and 32% decrease in their LDL and
total cholesterol levels, respectively [54]. The result by
Anderson and Gilliland [87] showed a significant decrease
(2.4%) in blood cholesterol for fermented milk containing L.
acidophilus during controlled clinical trials. In a randomized
clinical trial on yoghurt starters plus Bifidobacterium longum,
participants showed a decrease in total cholesterol [50]. In a
randomized, double-blind, placebo-controlled clinical trial,
it was demonstrated that E. faecium probiotic strain reduced
cholesterol levels by 12%. Klein et al. [56] also observed
a significant reduction (11.6%) in serum triglyceride levels
during the period probiotics consumption in a placebo-
controlled, double-blind, randomized crossover study. More
recently,Jonesetal.[57] reported that yoghurt formulation
containing microencapsulated bile salt hydrolase- (BSH-)
active Lactobacillus reuteri NCIMB 30242 is efficacious and
safe for lowering LDL-C, TC, apoB-100, and non-HDL-C
in hypercholesterolaemic subjects. Altogether, findings from
in vitro systems and animal studies as well as human trails
strongly suggest that probiotics have potential to ameliorate
the cholesterol metabolic dysfunction, especially mediated
through BSH activity and other unknown mechanisms, but
the exact mechanism(s) of action for probiotics’ mediated
decrease in cholesterol levels are not completely known.
Here, before discussing the probiotics’ mechanism of action
on plasma lipids, a summary of lipoprotein synthesis and
metabolism is reviewed, as follows for better understanding
by general readers the point of actions of probiotics on
lipid/cholesterol metabolism.
4.1. Plasma Lipoprotein Synthesis and Metabolism. Impor-
tant organs in the body that are responsible for synthesis
and transport of lipoprotein are the liver and the gut. A
cystic duct brings bile from the gallbladder to the gut. The
liver produces the bile, but it is moved to the gallbladder
and remains there to be used. Once a fatty meal arrives
at the small intestine, bile salts get into action and help
with emulsification of the fats. This makes their digestion
and absorption in the gut possible. Fatty acids, triglycerides,
and cholesterol combine in the epithelial cells of the gut
where they are covered with a layer of protein. These are
called chylomicrons [88]. The lymphatic system absorbs
these chylomicrons and later releases them into the blood.
Chylomicrons find their way to the liver and it turns them
into triglyceride and cholesterol. Bile salts do not end up in
the gut with the fats. They move down all the way to the
ileum, where, most of the bile salts are absorbed once again
and entered into the blood. The circulation takes the bile salts
back to the liver. They remain in the gallbladder, with bile to
be used for the above process again. Some of the bile salts are
not absorbed in the small intestine and end up in the colon
and are disposed of with feces. The liver makes up for the
loss of bile salts by synthesizing them from its cholesterol
reservoir. Cells in the liver also synthesize cholesterol and
are therefore another major source of the body’s cholesterol
pool, in addition to the dietary sources of cholesterol. A
number of factors, such as genes and diet, modulate the liver
to produce cholesterol.
4.1.1. Biosynthesis of Cholesterol. Just less than 50% of the
body’s cholesterol comes from new biosynthesis, nearly 10%
in the liver and 15% in the intestine [88]. Cholesterol syn-
thesis occurs in the microsomes and cytoplasm from the two-
carbon acetate group of acetyl-CoA [89]. The biosynthesis of
cholesterol goes through the following stages [89]:
(i) conversion of acetyl-CoAs to 3-hydroxy-3-methyl-
glutaryl-CoA (HMG-CoA),
(ii) conversion of HMG-CoA to mevalonate,
(iii) changing of mevalonate to isopentenyl pyrophos-
phate,
(iv) changing of isopentenyl pyrophosphate to squalene,
(v) conversion of squalene to cholesterol.
4.1.2. Regulating Cholesterol Synthesis. In healthy adults,
about 1 gram of cholesterol is synthesized and 0.3 gram is
consumed per day. The body maintains a relatively constant
amount of cholesterol (150–200 mg/dL). This is done mainly
through controlling the level of de novo synthesis. Dietary
intake of cholesterol in part regulates the level of cholesterol
synthesis. Both of these cholesterols are then used in the
formation of membranes and in the synthesis of the steroid
hormones and bile acids [90]. Bile acid synthesis uses most
of this cholesterol.
Three separate mechanisms regulate the body’s constant
supply of cholesterol from cells [88]asfollows
(i) regulation of HMG-CoA reductase (HMGR),
(ii) regulation of extra intracellular free cholesterol via
acyl-CoA cholesterol acyltransferase (ACAT),
(iii) regulation of cholesterol levels in plasma via HDL-
mediated reverse transport and LDL receptor-me-
diated uptake,
The cholesterol pool of the liver is used in two important
ways. The liver utilizes part of it to produce bile salts, to be
stored in the gallbladder as a part of the bile and ends up in
the gut. There, the bile salts are involved in the emulsification
of fats and their ingestion and absorption. The rest of the
cholesterol is used for other requirements of the body. To
do this, the liver combines cholesterol from its pool with
triglycerides and covers it with a particular protein so that
it could be dissolved in the blood. These are somewhat large
molecules, known as VLDL (very-low-density lipoproteins).
The liver then drains them into the blood. Lipoprotein lipase
(LPL) exists in abundance all over the body, especially in the
walls of the arteries. This enzyme is involved in removing
triglycerides from VLDL cholesterol. In the process, the
VLDL shrinks in size and a relatively larger portion of it is
made up of what is called intermediate-density lipoproteins,
or IDL.

8Experimental Diabetes Research
Low-Density Lipoprotein (LDL). As the process continues
and more triglycerides are taken away, what is left is a dense
molecule referred to as low-density lipoprotein (LDL). This
lipoprotein still maintains a large amount of cholesterol.
The protein layer allows the tissues to use this cholesterol,
LDL receptors on these tissues that make this interaction
possible. In the tissues such as those of the liver and the
inner layer of the arterial wall, cholesterol is taken away
from low-density lipoproteins. Free radicals in the body
are very reactive and oxidative compounds that can oxidize
low-density lipoprotein cholesterol and help atherosclerotic
plaque to form in the arteries. Antioxidants in the body can
inhibit this process [91].
High-Density Lipoprotein (HDL). The liver also produces
another type of lipoprotein, named high-density lipoprotein.
This is different from VLDL, which is also produced in the
liver. It has little triglyceride and cholesterol and has a partic-
ular protein covering. High-density lipoprotein collects the
surplus cholesterol that cholesterol metabolizing cells cannot
utilize. Lecithin-cholesterol acyltransferase is an enzyme that
is responsible for transporting surplus cholesterol back to
HDL molecules. Unused cholesterol from arteries, liver,
and other tissues is absorbed by HDL cholesterol. There is
evidence that even some oxidized LDL can be removed by
the LCAT and HDL cholesterol [92]. As HDL circulates in
the body and collects the cholesterol from tissues, it becomes
mature and goes back to the liver. There, it is identified by its
lipoprotein covering and is lodged in the liver’s cholesterol
pool.
Apo-A-1. Apo-A-1 is the main apolipoprotein in HDL
cholesterol and performs a key function of collecting surplus
cholesterol from the outer cells and transporting it back
to the liver. It also has antioxidant and anti-inflammatory
properties [93].
Apo33-B/Apo-A ratio is an indicator of cardiovascular
risk. The higher the ratio, the higher the probability of
cholesterol deposits in the walls of the arteries [94].
Apo-B. Apo-B is found in all of the atherogenic particles;
VLDL, IDL, as well as large and small dense LDL cholesterol.
They all have one Apo-B molecule inside them. The number
of Apo-B, therefore, is an indicator of the number of the
above particles. Apo-B helps to capture these particles from
the walls of the arteries. On the other hand, the Apo-B
formed in the liver helps with stabilization and transfer of
cholesterol and triglycerides in plasma IDL, VLDL, and sd-
LDL, and with the collecting of cholesterol in the liver and
the outer tissues. Of all the Apo-B particles in the blood,
over ninety percent are in low-density-lipid cholesterol.
Low-to-normal LDL cholesterol may indicate an increase in
highly atherogenic sd-LDL particles that are readily oxidized,
leading to increased formation of plaques on the arteries
walls. Apo-B/Apo-A ratio is an indicator of cardiovascular
risk. The higher the ratio, the higher the probability of
cholesterol deposits in the walls of the arteries [94].
4.2. Probiotics’ Mechanism of Action on Lipids. It has been
proposed that, when probiotics settle in the gut, they
ferment indigestible carbohydrate from food. Their action
raises the short-chain fatty acids (SCFAs) in the gut [86].
SCFAs are produced from peptide, polysaccharide, protein,
and oligosaccharide, mainly by anaerobic bacteria, and are
the final product of bacteria’s activity into the GI tract.
In terms of quantity, carbohydrates are the main source
of short-chain fatty acids [86]. These large molecules get
depolymerised by a variety of hydrolytic enzymes that are
produced by bacteria and allow the organisms to ferment
their sugar content. SCFA can lower the lipids in blood
through blocking synthesis of hepatic cholesterol and/or
through redirecting plasma cholesterol toward the liver [95].
A hundred to 450 mmol of the SFCA is produced in the
large intestine every day with relative proportions of acetate,
propionate, and butyrate being about 60 : 20 : 15 depending
on the substrate [86]. While acetate seems to increase total
cholesterol, propionate increases glucose in the blood and
reduces hypercholesterolemia response caused by acetate.
Propionate does that by decreasing its use by the liver, for
cholesterol and fatty acids synthesis. In addition, SCFAs
are potential modulator of food intake and energy sensing
process into the brain, which might indirectly play an impor-
tant role in reduction of cholesterol and other metabolism
deranging lipids into the host body [96]. Micelles, which
play a role in the absorption of cholesterol in the intestine,
are produced by bile salts, cholesterol, and phospholipids.
By producing bile acids through deconjugating the bile salts
in the small intestine, probiotics prevent micelle production.
When cholesterol enters the enterohepatic circulation, it is
death in the same way. Probiotics by using hydroxysteroid
dehydrogenase, and conjugated bile acid hydrolase enzymes,
breakdown the bile acid and hydrolyze bile salts. By doing so
bile acids’ enterohepatic circulation will be disrupted [97–
99]. Hydroxymethylglutarate CoA (HMG CoA) is another
compound that helps probiotics block HMG-CoA reductase
activity, which is a rate-limiting enzyme and is involved
in endogenous production of cholesterol. Probiotic bacteria
reduce absorption of cholesterol in the intestine by binding
and hence incorporating it to the cell membrane. Cholesterol
can also be assimilated during growth [100]. All of the above-
mentioned activities together help with the cholesterol-
lowering actions of probiotics.
4.3. Mechanisms of Cholesterol-Lowering Effects. Past in vitro
studies have evaluated a number of mechanisms proposed
for the cholesterol-lowering effects of probiotics and pre-
biotics. One of the purported mechanisms includes enzy-
matic deconjugation of bile acids by bile salt hydrolase of
probiotics. Bile, a water-soluble end product of cholesterol
in the liver, is stored and concentrated in the gallbladder
and released into the duodenum upon ingestion of food
[101]. It consists of cholesterol, phospholipids, conjugated
bile acids, bile pigments and electrolytes. Once deconjugated,
bile acids are less soluble and absorbed by the intestines,
leading to their elimination in the feces. Cholesterol is used
to synthesize new bile acids in a homeostatic response,

Experimental Diabetes Research 9
resulting in lowering of serum cholesterol [101](Figure 1).
In an in vitro study, Jones et al. [102] evaluated the
role of bile salt hydrolase in cholesterol lowering using
Lactobacillus plantarum. The authors found that BSH activity
was able to hydrolyze conjugated glycodeoxycholic acid and
taurodeoxycholic acid, leading to the deconjugation of glyco-
and taurobile acids. The hypocholesterolemic effect of the
probiotics has also been attributed to their ability to bind
cholesterol in the small intestines. Usman [27] previously
reported that strains of Lactobacillus gasseri could remove
cholesterol from laboratory media via binding onto cellular
surfaces. The ability of cholesterol binding appeared to
be growth and strain specific. Kimoto et al. [103]later
strengthened such a hypothesis by evaluating the removal
of cholesterol by probiotics cells during different growth
conditions. Live and growing cells were compared to those
that were nongrowing (live but suspended in phosphate
buffer) and dead (heat-killed). It was observed that, although
growing cells removed more cholesterol than dead cells, the
heat-killed cells could still remove cholesterol from media,
indicating that some cholesterol was bound to the cellular
surface. Cholesterol was also removed by probiotics by
incorporation into the cellular membranes during growth.
Kimoto et al. [103] have examined the removal of cholesterol
by several strains of lactococci from media. A difference in
the fatty acid distribution pattern was observed for cells
grown in the presence and absence of cholesterol. Lipids
of probiotics are predominantly found in the membrane,
suggesting that cholesterol incorporated into the cellular
membrane had altered the fatty acid composition of the cells.
The incorporation of cholesterol into the cellular membrane
increased the concentration of saturated and unsaturated
fatty acids, leading to increased membrane strength and
subsequently higher cellular resistance toward lysis [104,
105]. Lye et al. [104] also further evaluated this mechanism
by determining the possible locations of the incorporated
cholesterol within the membrane phospholipid bilayer of
probiotic cells. Fluorescence probes were incorporated into
the membrane bilayer of probiotic cells that were grown
in the absence and presence of cholesterol. Enrichment of
cholesterol was found in the regions of the phospholipid tails,
upper phospholipids, and polar heads of the cellular mem-
brane phospholipid bilayer in cells that were grown in the
presence of cholesterol compared to the control cells, indicat-
ing incorporation of cholesterol in those regions. Cholesterol
can also be converted in the intestines to coprostanol, which
is directly excreted in feces. This decreases the amount of
cholesterol being absorbed, leading to a reduced concentra-
tion in the physiological cholesterol pool. Possible conversion
of cholesterol into coprostanol by bacteria has been evaluated
by Chiang et al. [106]. In their study, it was found that
cholesterol dehydrogenase/isomerase produced by bacteria
such as Sterolibacterium denitrificans was responsible for
catalyzing the transformation of cholesterol to cholest-4-
en-3-one, an intermediate cofactor in the conversion of
cholesterol to coprostanol. This served as a fundamental for
further evaluations using strains of probiotic bacteria. In a
recent in vitro study, Lye et al. [105] evaluated the conversion
of cholesterol to coprostanol by strains of lactobacilli such
as Lactobacillus acidophilus, L. bulgaricus, and L. casei
ATCC 393 via fluorometric assays. The authors detected
both intracellular and extracellular cholesterol reductase
in all strains of probiotics examined, indicating possible
intracellular and extracellular conversion of cholesterol to
coprostanol. The concentration of cholesterol in the medium
also decreased upon fermentation by probiotics accompa-
nied by increased concentrations of coprostanol. This mech-
anism warrants further evaluations as cholesterol reductase is
also directly administered to humans to convert cholesterol
to coprostanol in the small intestines for a bloodstream
cholesterol-lowering effect. Most of the hypotheses raised to
date are based on in vitro experiments, and few attempts
have been made to evaluate the possible hypocholesterolemic
mechanisms based on in vivo trials. Most of the in vivo
trials conducted thus far have focused heavily on verifying
the hypocholesterolemic effects of probiotics, rather than the
mechanisms involved. Liong et al. [107] had evaluated the
hypocholesterolemic effect of a synbiotic and the possible
mechanisms involved by using hypercholesterolemic pigs.
In their parallel 8-week study, the authors found that
the administration of a synbiotic containing L. acidophilus
ATCC 4962, fructooligosaccharides, inulin, and mannitol
decreased plasma total cholesterol, LDL-cholesterol, and
triacylglycerols compared to the control. These lipoproteins
were subsequently subfractionated and characterized. Pigs
supplemented with the synbiotic had a lower concentration
of cholesteryl esters in the LDL particles, accompanied by
a higher concentration of triacylglycerol. Triacylglycerol-
enriched LDL particles are more susceptible to hydrolysis
and removal from blood, while loss of cholesteryl esters
forms smaller and denser LDL particles leading to a higher
removal from blood compared to larger LDL particles. The
authors also found that the administration of the synbiotic
led to higher concentration of cholesteryl esters in the
HDL particles. HDL is termed as the beneficial cholesterol
attributed to its role of transporting cholesterol to the
liver for further hydrolysis. Cholesterol is transported as
cholesteryl esters in the core of HDL. Thus, it was suggested
that the synbiotic induced a hypocholesterolemic effect via
altering the pathways of cholesteryl esters and lipoprotein
transporters. Prebiotics such as inulin and fructooligosac-
charides are soluble, indigestible, viscous, and fermentable
compounds that contribute to hypocholesterolemia via two
mechanisms: decreasing cholesterol absorption accompa-
nied by enhanced cholesterol excretion via feces and the
production of short-chain fatty acids (SCFAs) upon selective
fermentation by intestinal bacterial microflora (Figure 2)
[108]. Using hypercholesterolemic-induced rats, Kim and
Shin [109] also found that the administration of inulin for
4-weeks decreased serum LDL-cholesterol with increased
serum HDL-cholesterol levels (P<0.05) compared to the
control. Rats fed with inulin also showed higher excretions
of fecal lipid and cholesterol compared to the control (P<
0.05), mainly attributed to reduced cholesterol absorption.
Similar to indigestible fibers, soluble indigestible prebiotics
have been postulated to increase the viscosity of the digestive
tract and increase the thickness of the unstirred layer in the
small intestine and thus inhibiting the uptake of cholesterol

10 Experimental Diabetes Research
[110]. This may have led to a higher cholesterol catabolism
in the liver that contributed to a hypocholesterolemic effect.
5. Conclusion and Future Prospects
Probiotics have received much attention on their proclaimed
health benefits which include improvement in lactose intol-
erance, increase in natural resistance to infectious disease
in gastrointestinal tract, suppression of cancer, reduction in
serum cholesterol level, and improved digestion. In addition,
there has been considerable interest in the effect of probiotics
on human lipid metabolism, and numerous studies have
focused on the potential hypocholesterolemic activity of
probiotics in human. Despite these claimed benefits from
the human clinical studies carried out for the last two
decades, a decisive outcome has failed to be reached due
to controversies raised. Also, the exact mechanism for
cholesterol removal is poorly understood. Several possible
mechanisms for cholesterol removal by probiotics have been
proposed including assimilation of cholesterol by growing
cells, binding of cholesterol to cellular surface, incorporation
of cholesterol into the cellular membrane, deconjugation of
bile via bile salt hydrolase, and coprecipitation of cholesterol
with deconjugated bile; however, some of these mechanisms
are strain dependent, and conditions generated under lab-
oratory conditions would not be practical in the in vivo
systems. Such discrepancies in the data of different effects
on serum cholesterol levels may come from the differences
in genus, species, and strains of lactic acid bacteria. Even
though the hypocholesterolemic mechanism of probiotics
has not yet been fully understood, it is an established fact
that cholesterol and bile salt metabolism are closely linked.
Recently “BSH hypothesis” has being proposed to explain
cholesterol-lowering effects of probiotics. More recently,
the hypocholesterolemic effects of some probiotics, which
showed high BSH activities from in vitro trials, have been
confirmed in human as well as in animals. However, the
hypocholesterolemic mechanism of probiotics based on the
BSH hypothesis has not yet been sufficiently elucidated.
Moreover, considering that a number of commercial pro-
biotic strains exhibit high BSH activities, further studies
are needed to determine whether the BSH activity of the
probiotics strains is beneficial or detrimental to the host. In
probiotic research, bile tolerance is considered of primary
importance in the selection of strains as bile tolerance enables
the bacteria to survive its transit along the duodenum and
subsequently to grow and colonize the gut epithelia. Thus, it
is important to understand the physiological and molecular
mechanisms by which enteric microorganisms including
bifidobacteria have evolved to resist against antimicrobial
activity of bile in the GI tract. Further investigation on the
conserved and variable regions of the bsh genes from various
species could be useful for the development of alternative
phylogenetic marker for bifidobacteria. Furthermore, one of
the future challenges will be to unravel the physiological
impacts of bile salt hydrolase activity on the enzyme-
producing bacterial and mammalian cells.
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