Petra Olejníková1, Martin Šimkovič1, Boris Lakatoš1, Lucia Birošová2, Barbora Kaliňáková1, Pavol Olejník3, Alexander Kaszonyi4
1Oddelenie
biochémie a mikrobiológie, FCHPT STU, Radlinského 9, 812 37 Bratislava,e-mail: petra.olejnikova@stuba.sk, 2Oddelenie výživy a ochrany zdravia, FCHPT STU,
Radlinského 9,812 37 Bratislava,3 Oddelenie manažmentu, STU, Vazovova 5, 812 43 Bratislava 34 Oddelenie organickej technológie FCHPT STU, Radlinského 9,812 37 Bratislava
The intestinal microflora can be moderated by the dietary intake. It consists of cca. 50 bacterial species and contribute to the well being of humans. The main baceterial species making up the colon microflora
belong to anaerobes and facultative anaerobes such as Enterobacteriaceae, Bacteroides, Clostridia, Veiolonella, Bifidobacteria and Lactobacilli. The food intake and diet has a special impact on intestinal flora and
thereby is able to affect the human health status. One of the health risks parameter is the cholesterol pool. Endogenous cholesterol is synthesized mainly in the liver and the small intestine, and the exogenous
source is mainly of dietary origin. Cholesterol in the intestine can be reabsorbed, but there are also some microbes that are able to convert cholesterol by oxidation and hydrogenation into coprostanol (5cholestane-3-ol) via coprostanone (5-cholestane-3-one) to coprostanol, unabsorbable, and thus reducing the intestinal cholesterol pool. Minor products of cholesterol transformation are cholestanol (5-cholestane-3ol) and cholestanone (5-cholestane-3-one). Furthermore, phytosterols such as sitosterol (24-ethyl-cholest-5-ene-3-ol) and campesterol (24-methyl-cholest-5-ene-3-ol) are predominantly converted to the analogous
5-cholestanes by colonic bacteria. The present study was designed to compare further the number and type of microorganisms making up the fecal microflora of strict vegetarians and persons eating normal middle
European diet and to find a crosslink between colonal microflora and the sterol pool in the feces.
The subjects studied consisted of 240 healthy
persons from Slovakia; 106 non-vegetarians and
86 semivegetarians and 56 lactoovovegetarians
ranged in age from 20 to 60 years. Fresh fecal
specimens were collected in tubes, were
transported at 4°C and were immediately
processed. Samples approximately 1 g [wet
weight] were suspended in Brain Heart Infusion
and serial 10-fold dilutions from 10-1 to 10-8
were prepared. The method for bacterial
analysis of the fecal microflora in this study
was that of Mitsuoka et al. (1) For the bacterial
species number, the bacterial CFU per gram of
wet feces were calculated and converted into a
logarithmic equivalent. The fecal sterols were
assayed according to Keller and Jahreis and
analysed by GC-MS method.(2)
Comparison of fecal microbiota
The same genera of strict anaerobes, facultative anaerobes and aerobes, were present in the feces of each group of volunteers (Table 1). Strict anaerobes
belonged to the genera Clostridium, Bacteroides, Veilonella, Bifidobacterium and Lactobacillus. The facultative anaerobes and aerobes present in the feces
belonging to the genera Escherichia, Enterobacter, Citrobacter, and Proteus, Enterococcus and Staphylococcus. The most prevalent facultative anaerobes
were Escherichia coli, Enterobacter cloace; Enterobacter aerogenes and the H2S producing Citrobacter farmeri. Some yeasts and fungi were found in feces of
semivegetarians, non-vegetarians and lactoovovegetarians (CFUlog10= 3,6-4,2). One fungal isolate was able to grow in anaerobic and also in aerobic conditions.
According its ITS sequence it belonged to the genera Aspergillus sp.
Tab.1 Comparison of fecal microflora of vegetarians semivegetarians and non-vegetarians (log10 CFU/g feaces)
microbial groups
Fecal sterols content
The total sterol content in feces of semivegetarians was
amounted to 54.71 ± 9.1 mg/g dry feces. This figured the
highest sterol value compared with non-vegetarians and
lactoovovegetarians. The lowest sterol value was observed in
the group of lactoovovegetariants 14.13 ± 5,5 mg/g dry
feces. A wide inter-individual range in the conversion of
cholesterol into degradation products (coprostanol,
coprostanone, cholestanol) was observed - conversion rate:
0,23–99%).
total facultative anaerobes
total anaerobes
yeasts and fungi
Clostridium sp.
lecitinase-positive Clostridium sp.
Bacteroides sp.
Veilonella sp.
spores
Bifidobaceteria+Lactobacilli
Enterococci
Enterobacteriace
Styphylococcus aureus
coprostanol
18%
sitosterol
12%
coprostanon
8%
cholesterol
15%
campestreol
7%
metylcoprostanon
9%
metylcoprostanol
6%
Fig.1 The sterol content of nonvegetarians
Sterol content of semivegetarians
stigmasterol
9%
coprostanol
15%
sitosterol
13%
coprostanon
9%
etylcoprostanol
11%
cholesterol
12%
cholestenon
8%
campestreol
7%
metylcoprostanol
7%
metylcoprostanon
9%
Fig.2 The sterol content of semivegetarians
Sterol content of
lactoovovegetarians
stigmasterol
1%
sitosterol
22%
coprostanol
26%
etylcoprostanol
9%
cholestenon
0,1%
coprostanon
4%
non-campestreol
metylcoprostanon
5%
metylcoprostanol
1%
cholesterol
32%
8,7
8,6
3,6
8,4
1,0
3,0
7,7
0,93
6,4
7,1
8,0
1,0
0,13
0,18
0,23
0,25
0,21
0,26
0,20
0,18
0,24
0,21
0,16
0,21
SE
8,5
8,3
4,2
8,8
0,66
3,2
7,9
1,1
6,9
6,7
7,7
0,96
0,19
0,23
0,4
0,15
0,28
0,32
0,29
0,30
0,19
0,44
0,18
0,30
nonvegetarians
8,3
8,4
3,8
8,6
1,3
3,2
7,1
0,86
6,1
6,6
7,4
1,0
SE
0,17
0,17
0,17
0,13
0,21
0,21
0,22
0,15
0,21
0,17
0,16
0,19
The microbial community in the human colon contains cholesterol-to-coprostanol reducing bacteria but all of
the species responsible for this conversion are still unknown. A cholesterol-reducing bacterium
of human intestinal origin was described in some papers. It belongs to the bacterial group Bacteroidetes and
was described as Eubacterium coprostanoligenes (3,4). In some works are lactobacilli described as bacteria
that are able to reduce cholesterol via conversion of cholesterol to coprostanol, aided by the ability of strains
to produce cholesterol reductase and could remove cholesterol via the incorporation of cholesterol into the
cellular membrane. (5) In our study we made a correlation between the level of cholesterol conversion to
coprostanol and the fecal flora. A good correlation was found between the fecal content of lactobacilli +
bifidobaceria. The range of conversion coefficient was individual for each (0,23 to 99%). The correlation
between higher content of lactobacilli and bifidobacteria and high conversion coefficient was found (Tab.2)
stigmasterol
8%
cholestenon
7%
SE
Lactoovovegetarians
Microbial conversion of cholesterol to coprostanol
Sterol content of
nonvegetarians
etylcoprostanol
10%
Semi
vegetarians
Fig.4 Predicted mechanism of microbial cholesterol conversion
Tab.2 The correlation between cholesterol conversion and microbiota
(high convertors)
cholesterol
convertion
to coprotanol (%)
98,37
98,17
98,74
99,57
99,50
90,76
90,26
90.50
95,52
94,87
96,69
lactobacilli
+bifidobacteria
(CFU/g feces)
1,00E+10
1,00E+09
1,00E+07
1,00E+07
1,00E+08
1,00E+08
1,00E+09
1,00E+07
1,00E+08
1,00E+08
1,00E+08
Tab.3 The correlation between cholesterol conversion and microbiota
(low convertors)
cholesterol
convertion to
coprotanol (%)
1,80
0,76
lactobacilli
+bifidobacteria
(CFU/g feces)
1,00E+06
1,00E+05
0,23
1,00E+03
1 . Mitsuoka T., K. Ohno, Y. Benno, K. Suzuki, and K. Namba.: Die Faekalflora bei Menschen. V. Mitteilung: Vorgleich des neu
entwickelten Verfahrens mit dem bisherigen ublichen Verfahren zur Darmfloraanalyse. Zentralbl. Bakteriol. Mikrobiol. Hyg. 1
Abt Orig. A 234:219-233, 1976
2. Keller S., Jahreis G.:Determination of underivatised sterols and bile acid trimethyl silyl ether methyl esters by gas
chromatography–mass spectrometry–single
ion monitoring in faeces, J Chromatography B, 813, 199–207, 2004
3. Lye H S., Rusul G, Liong MT: Removal of cholesterol by lactobacilli via incorporation and conversion to coprostanol., J. Diary
Sci., 93(4):1383-92, 2010
4. Gerard P, Leperca P, Leclerc M, Gavini F, Raibaud P, Juste C: Bacteroides sp. strain D8, the first cholesterol-reducing
bacterium isolated from human feces. 2007 Sep;73(18):5742-9. Epub 2007 Jul 6.
5. Norin E.:Intestinal Cholesterol Conversion in Adults and Elderly from Four Different European Countries, Ann Nutr Metab
52(suppl 1):12–14,2008
.
Fig.3The sterol content of lactoovovegetarians
This work was made during realisation of the project „Výskum zdravotných efektov rastlinnej potravy a možnosti redukcie zdravotných rizík“ supported by the European fond for regional development. ( ITMS kód: 26240220022)