CHOLESTEROL ASSIMILATION BY COMMERCIAL YOGHURT STARTER CULTURES

RUM POLO
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S ACTA Acta Sci. Pol., Technol. Aliment. 6(1) 2007, 83-94

CHOLESTEROL ASSIMILATION BY COMMERCIAL
YOGHURT STARTER CULTURES
Ma?gorzata Ziarno, Ewa S?kul, Alvaro Aguado Lafraya
Warsaw Agricultural University SGGW
Abstract. The ability to in vitro cholesterol level reduction in laboratory media has been
shown for numerous strains of lactic acid bacteria, but not for all strains of lactic bacteria
used in the dairy industry. The aim of this work was the determination of the ability of se-
lected thermophilic lactic acid bacteria to cholesterol assimilation during 24 h culture in
MRS broth. Commercial starter cultures showed various ability to cholesterol assimilation
from laboratory medium. In case of starter cultures used for production of traditional yo-
ghurt, consisting of Streptococcus salivarius subsp. thermophilus and Lactobacillus del-
brueckii subsp. bulgaricus, the quantity of assimilated cholesterol did not exceed 27% of
its initial contents (0.7 g in 1 dm3). Starter cultures used for bioyoghurt production, con-
taining also probiotic strains (came from Lactobacillus acidophilus species or Bifidobac-
terium genus) assimilated from almost 18% to over 38% of cholesterol. For one monocul-
ture of Lb. acidophilus, cholesterol assimilation ability of 49-55% was observed, despite
that the number of bacterial cells in this culture was not different from number of bacteria
in other cultures.
Key words: cholesterol, dairy starter cultures, assimilation, lactic acid bacteria
INTRODUCTION
The ability to in vitro cholesterol level reduction in model culture media has been
shown for numerous strains of lactic acid bacteria, such as Lb. acidophilus, Lb. del-
brueckii subsp. bulgaricus, Lb. casei, Lb. gasseri, Lb. amylovorus [Gilliland et al. 1985,
Rasic et al. 1992, Walker and Gilliland 1993, Buck and Gilliland 1994, Tahri et al.
1996, Noh et al. 1997, Brashears et al. 1998, Grill et al. 2000, Lin and Chen 2000,
Hosono et al. 2002]. Other species of bacteria in which a similar activity was observed
are: Bifidobacterium (B. bifidum, B. longum), Streptococcus (Str. salivarius subsp.
thermophilus) and Enterococcus (E. faecium), as well as Lactococcus (Lc. lactis subsp.
lactis, Lc. lactis subsp. lactis var. diacetylactis), Leuconostoc mesenteroides subsp.
mesenteroides [Rasic et al. 1992, Hosono and Tono-Oka 1995, Tahri et al. 1996, Tar-
anto et al. 1996, Dambekodi and Gilliland 1998, Kimoto et al. 2002]. Adhesion of the

Corresponding author – Adres do korespondencji: Dr in . Ma?gorzata Ziarno, Department of
Biotechnology, Microbiology and Food Evaluation of Warsaw Agricultural University SGGW,
Nowoursynowska 159 C, 02-776 Warsaw, Poland, e-mail: malgorzata_ziarno@sggw.pl

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84
M. Ziarno ...
cholesterol to the cell surface and incorporation of the cholesterol into the cellular
membrane are the most frequently suggested mechanisms of lactic bacteria activity on
cholesterol level [Hosono and Tono-Oka 1995, Noh et al. 1997, Brashears et al. 1998,
Dambekodi and Gilliland 1998]. This raised an assumption that similar assimilation
phenomenon may also take place in dairy products fermented by lactic bacteria. This
seems to be a proof that both probiotic, and traditional strains of lactic acid bacteria
included in starter cultures used for production of fermented dairy products possess
ability to cholesterol assimilation. However, not all strains of lactic bacteria used in
dairy industry have been studied in this respect.
The aim of this work was the determination of the ability of selected thermophilic
lactic acid bacteria to cholesterol assimilation during 24 h culture in MRS broth.
MATERIALS AND METHODS
Starter cultures and culture conditions. Five commercial single species and 13
multiple species lyophilized starter cultures used in dairy industry for production of
yoghurts and bio-yoghurts were used in this study. Seven first thermophilic cultures
contained Str. salivarius subsp. thermophilus and Lb. delbrueckii subsp. bulgaricus
(Table 1, symbols A-G). Six next cultures were the mixtures of Bifidobacterium and other
Table 1. The results of quantitative analysis of culture of traditional yoghurt starter cultures (mean
log10 ±standard deviation)
Tabela 1. Wyniki analizy liczby bakterii w tradycyjnych kulturach jogurtowych (?rednia warto??
logarytmu i odchylenie standardowe)
Log10 of number
Log10 of number
of thermophilic lactobacilli
of thermophilic streptococci
Starter
in 1 cm3
in 1 cm3
culture
Qualitative constitu-
Logarytm liczby termofilnych
Logarytm liczby termofilnych
symbol tion of starter culture
pa?eczek
streptokoków
Symbol
Jako?ciowy sk?ad
kultury
kultury starterowej
MRS without
MRS with
M17 without
M17 with choles-
startero-
cholesterol
cholesterol
cholesterol
terol
wej
MRS bez
MRS
M17 bez
M17
cholesterolu
z cholesterolem
cholesterolu
z cholesterolem
A
Str. salivarius
8.4 ±0.40 a
8.4 ±0.35 a
8.6 ±0.22 A
8.5 ±0.13 A
subsp. thermophilus,
B
8.9 ±0.47 a
8.8 ±0.40 a
–
–
Lb. delbrueckii
C
subsp. bulgaricus
8.4 ±0.44 a
8.5 ±0.31 a
6.8 ±0.66 A
6.9 ±1.05 A
D
8.5 ±0.36 a
8.5 ±0.16 a
–
–
E
8.2 ±0.29 a
8.4 ±0.35 a
–
–
F
8.4 ±0.32 a
8.3 ±0.50 a
–
–
G
7.7 ±0.31 a
7.7 ±0.22 a
7.5 ±0.54 A
7.5 ±0.32 A
– not measured.
a, b, c, ..., A, B, C, ... – means from the same row, with different superscripts, are significant different at
? = 0.05.
– nie mierzono.
a, b, c, ..., A, B, C, ... – ró ne indeksy przy ?rednich z tego samego wiersza oznaczaj? statystycznie istotna
ró nic? mi?dzy tymi ?rednimi dla ? = 0,05.
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Cholesterol assimilation by commercial yoghurt starter cultures
85
thermophilic lactic acid bacteria (Table 2 and 3, symbols H-M). Last five cultures were
the following monocultures: Bifidobacterium bifidum (culture N in Table 3), Bifidobac-
terium species (culture O in Table 3), Lb. acidophilus (cultures P and Q in Table 3), Str.
salivarius subsp. thermophilus (culture R in Table 3).
Table 2. The results of quantitative analysis of culture of yoghurt starter cultures contained probi-
otic strains (mean log10 ±standard deviation)
Tabela 2. Wyniki analizy liczby bakterii w kulturach jogurtowych zawieraj?cych szczepy probio-
tyczne (?rednia warto?? logarytmu i odchylenie standardowe)
Log10 of number
Log10 of number
of thermophilic lactobacilli
of thermophilic streptococci
Starter
in 1 cm3
in 1 cm3
culture
Qualitative constitution
Logarytm liczby termofilnych
Logarytm liczby termofilnych
symbol
of starter culture
pa?eczek
streptokoków
Symbol Jako?ciowy sk?ad kultury
kultury
starterowej
MRS without
MRS with
MRS without
MRS with
startero-
cholesterol
cholesterol
cholesterol
cholesterol
wej
MRS bez
MRS
MRS bez
MRS
cholesterolu
z cholesterolem
cholesterolu
z cholesterolem
H
Str. salivarius
8.2 ±0.55 a
8.1 ±0.42 a
8.3 ±0.57 A
8.2 ±0.39 A
subsp. thermophilus,
Lb. acidophilus,
Bifidobacterium lactis
I
Str. salivarius
8.1 ±0.41 a
8.0 ±0.34 a
8.5 ±0.51 A
8.2 ±0.58 A
subsp. thermophilus,
J
8.0 ±0.50 a
7.9 ±0.47 a
8.3 ±0.61 A
8.0 ±0.44 A
Lb. delbrueckii
subsp. bulgaricus,
K
8.0 ±0.71 a
8.0 ±0.34 a
–
–
Lb. acidophilus,
Bifidobacterium species
L
Str. salivarius
8.0 ±0.57 a
7.8 ±0.68 a
8.0 ±0.43 A
7.9 ±0.64 A
subsp. thermophilus,
Lb. delbrueckii
subsp. bulgaricus,
Lb. acidophilus,
Bifidobacterium bifidum
– not measured.
a, b, c, ..., A, B, C, ... – means from the same row, with different superscripts, are significant different at
? = 0.05.
– nie mierzono.
a, b, c, ..., A, B, C, ... – ró ne indeksy przy ?rednich z tego samego wiersza oznaczaj? statystycznie istotna
ró nic? mi?dzy tymi ?rednimi dla ? = 0,05.
The cultures were grown in MRS broth (Merck) in 37°C for 24 hours, then were
transferred into a fresh MRS broth portion and were further grown in 37°C for 24 hours,
afterwards, then were used for study.
Cholesterol solution preparation. Cholesterol of chemical purity > 99% (Sigma-
Aldrich) was hot dissolved in 99% ethanol and Tween 80, mixed in 3:1 ratio. The ob-
tained solution had a concentration of 3.0 g of cholesterol in 1 dm3. This solution was
used in experiments concerning cholesterol assimilation from MRS broth by LAB. The
sterilely measured portion of this solution was added to MRS broth in such a quantity,
so that the final concentration of the cholesterol was 0.7 g in 1 dm3 of the basis.
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86
M. Ziarno ...
Table 3. The results of quantitative analysis of culture of single and multiple species starter cul-
tures of probiotic strains (mean log10 ±standard deviation)
Tabela 3. Wyniki analizy liczby bakterii w monokulturach i mieszankach kultur szczepów probio-
tycznych (?rednia warto?? logarytmu i odchylenie standardowe)
Log10 of number
Log10 of number
of thermophilic lactobacilli
of thermophilic streptococci
Starter
in 1 cm3
in 1 cm3
culture
Qualitative constitution
Logarytm liczby termofilnych
Logarytm liczby termofilnych
symbol
of starter culture
pa?eczek
streptokoków
Symbol Jako?ciowy sk?ad kultury
kultury
starterowej
MRS without
MRS with
MRS without
MRS with
startero-
cholesterol
cholesterol
cholesterol
cholesterol
wej
MRS bez
MRS
MRS bez
MRS
cholesterolu
z cholesterolem
cholesterolu
z cholesterolem
M
Lb. rhamnosus,
8.2 ±0.72 a
7.8 ±0.64 a
–
–
Bifidobacterium species
N
Bifidobacterium bifidum
8.1 ±0.52 a
7.9 ±0.58 a
–
–
O
Bifidobacterium species
7.6 ±0.54 a
7.6 ±0.29 a
–
–
P
Lb. acidophilus
7.9 ±0.86 a
7.8 ±0.55 a
–
–
Q
7.8 ±0.49 a
7.8 ±0.27 a
–
–
R
Str. salivarius
–
–
8.1 ±0.54 A
7.8 ±0.36 A
subsp. thermophilus
– not measured.
a, b, c, ..., A, B, C, ... – means from the same row, with different superscripts, are significantly different at
? = 0.05.
– nie mierzono.
a, b, c, ..., A, B, C, ... – ró ne indeksy przy ?rednich z tego samego wiersza oznaczaj? statystycznie istotna
ró nic? mi?dzy tymi ?rednimi dla ? = 0,05.
Cholesterol assimilation from MRS broth assay. The revived starter cultures’
ability for cholesterol assimilation was calculated as loss of its concentration in MRS
broth supernatant after the end of culture in 37°C for 24 hours. Cholesterol concentra-
tion was assayed with the enzymatic diagnostic test Cholésterol RTU® (BioMérieux).
Absorbance was measured with a spectrophotometer SmartSpecTM 3000 (Bio-Rad).
Before cholesterol concentration in MRS broth was assayed the content of tubes was
centrifuged in ultra-speed centrifuge (12.000 g, 10 min, 4°C) in order to separate bacte-
rial cells biomass and obtain clear MRS broth supernatant. Percentage value of choles-
terol assimilation was calculated using the following formula
A – B
D = 100 ?
?100, %
C
where: A – cholesterol concentration in MRS broth containing suspension of bacterial
cells and addition of cholesterol solution,
B – cholesterol concentration in MRS broth containing bacterial cells suspen-
sion only,
C – cholesterol concentration in MRS broth containing cholesterol solution ad-
dition only,
D – cholesterol assimilation, %.
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Cholesterol assimilation by commercial yoghurt starter cultures
87
The assay was performed in five-fold repetition.
Number of lactic acid bacteria cells assay. The number of lactobacilli, bifidobac-
teria and streptococci was assayed with plate method in parallel cultures in MRS broth
containing or not containing an addition of cholesterol solution. Inoculated plates were
incubated in conditions optimal for a given species: for lactobacilli and bifidobacteria –
MRS Agar (Merck), 37°C for 72 hours, anaerobic conditions; for streptococci – M17
Agar (Merck), 37°C for 48 hours, aerobic conditions.
Statistical analysis. Results of assay of lactobacilli and streptococci numbers in
MRS broth containing and not containing the cholesterol solution were analysed using
Statgraphics 4.1 Plus software.
RESULTS
The studied commercial starter cultures showed various ability to assimilate choles-
terol from culture medium. The results of these studies are presented in Figures 1-3. In
the case of starter cultures used for production of traditional yoghurt, the quantity of
assimilated cholesterol did not exceed 27% of its initial contents. It is worth reminding
that such starter cultures contain only two species of thermophilic lactic acid bacteria:
Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bul-
garicus. For five such starter cultures average cholesterol assimilation value was from
almost 12% to over 21% (Fig. 1).

30.0

25.0
l
,

%

r
o
t
e
,

%
20.0
s
l
u
l
e
o
r
o
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t
e
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f

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l
e
15.0

o
o
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i
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i
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a
n
a
10.0
i
m
s
z
s
i
?

a
W
e
h
T
5.0
0.0
A
B
C
D
E
F
G
Starter culture symbol – Symbol kultury starterowej

Fig. 1. The assimilation of cholesterol by traditional yoghurt starter cultures
(means and standard deviations): A-G – symbols of cultures containing Str.
salivarius subsp. thermophilus and Lb. delbrueckii subsp. bulgaricus
Rys. 1. Wi?zanie cholesterolu przez tradycyjne jogurtowe kultury starterowe
(warto?ci ?rednie i odchylenia standardowe): A-G – symbole kultur zawiera-
j?cych Str. salivarius subsp. thermophilus i Lb. delbrueckii subsp. bulgaricus
Technologia Alimentaria 6(1) 2007

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88
M. Ziarno ...
This was confirmed also by results of cholesterol assimilation by starter cultures
used for bioyoghurt production. Besides two basic yoghurt bacteria species those cul-
tures contain also an additional microflora, for example Lactobacillus acidophilus spe-
cies or Bifidobacterium genus. Those starter cultures could assimilate cholesterol at the
level of almost 18% to over 38% (Fig. 2), which was significantly higher than for tradi-
tional yoghurt cultures.

45.0
40.0

35.0
l
,

%

r
o
t
e
,

%
30.0
s
l
u
l
e
o
r
o
h
t
e
s
25.0
f

c
l
e

o
o
n
h
20.0
t
i
o

c
i
e
i
l
a
n
i
m
a
15.0
s
z
s
i
?

a
W
e
10.0
h
T
5.0
0.0
H
I
J
K
L
Starter culture symbol – Symbol kultury starterowej

Fig. 2. The assimilation of cholesterol by yoghurt starter cultures containing
probiotic strains (means and standard deviations): H – symbol of culture
containing Str. salivarius subsp. thermophilus, Lb. acidophilus and Bifido-
bacterium lactis, I-K – symbols of cultures containing Str. salivarius subsp.
thermophilus, Lb. delbrueckii subsp. bulgaricus, Lb. acidophilus and Bifido-
bacterium species, L – symbol of culture containing Str. salivarius subsp.
thermophilus, Lb. delbrueckii subsp. bulgaricus, Lb. acidophilus and Bifido-
bacterium bifidum
Rys. 2. Wi?zanie cholesterolu przez jogurtowe kultury starterowe zawiera-
j?ce szczepy probiotyczne (warto?ci ?rednie i odchylenia standardowe): H –
symbol kultury zawieraj?cej Str. salivarius subsp. thermophilus, Lb. aci-
dophilus i Bifidobacterium lactis, I-K – symbole kultur zawieraj?cych Str.
salivarius subsp. thermophilus, Lb. delbrueckii subsp. bulgaricus, Lb. aci-
dophilus i Bifidobacterium species, L – symbol kultury zawieraj?cej Str. sa-
livarius subsp. thermophilus, Lb. delbrueckii subsp. bulgaricus, Lb. aci-
dophilus i Bifidobacterium bifidum
Analysis of monocultures or cultures composed of additional microflora only con-
firmed an assumption that those bacteria possess higher ability of cholesterol assimila-
tion comparing to traditional yoghurt cultures (Fig. 3). One monoculture of Lb. aci-
dophilus deserves a special attention. For this culture cholesterol assimilation ability of
49.2-55.4% was observed, despite the number of bacterial cells in this culture was not
different from number of bacteria in other cultures (Table 3). Comparably, the monocul-
ture of Str. salivarius subsp. thermophilus assimilated cholesterol at the level of 23.9%
±6.81% (Fig. 3).
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Cholesterol assimilation by commercial yoghurt starter cultures
89
60.0
55.0

50.0
l
,

%

45.0
r
o
t
e
,

%
s
l
u
40.0
l
e
o
r
o
h
t
e
35.0
s
f

c
l
e

o
o
30.0
n
h
t
i
o

c
i
e
25.0
i
l
a
n
i
m
a
z
20.0
s
s
i
?

a
W
15.0
e
h
T
10.0
5.0
0.0
M
N
O
P
Q
R
Starter culture symbol – Symbol kultury starterowej

Fig. 3. The assimilation of cholesterol by single and multiple species starter
cultures of probiotic strains (means and standard deviations): M – symbol of
culture containing Lb. rhamnosus and Bifidobacterium species, N – symbol
of monoculture of Bifidobacterium bifidum, O – symbol of monoculture of
Bifidobacterium species, P-Q – symbols of monocultures of Lb. acidophilus,
R – symbol of monoculture of Str. salivarius subsp. thermophilus
Rys. 3. Wi?zanie cholesterolu przez monokultury i mieszanki kultur szcze-
pów probiotycznych (warto?ci ?rednie i odchylenia standardowe): M – sym-
bol kultury zawieraj?cej Lb. rhamnosus i Bifidobacterium species, N – sym-
bol monokultury Bifidobacterium bifidum, O – symbol monokultury Bifido-
bacterium species, P-Q – symbol monokultury Lb. acidophilus, R – symbol
monokultury Str. salivarius subsp. thermophilus
At the same time it was checked if cholesterol solution addition inhibits or stimu-
lates growth of studied cultures. Logarithm of lactobacilli number in cultures was be-
tween 6.7 and 9.3, and of streptococci from 5.8 to 9.1. No statistically significant differ-
ences were observed both for number of streptococci and number of lactobacilli, be-
tween cultures carried in MRS broth with addition of cholesterol solution and without
this addition (Table 1-3). Found differences regarded the number of bacteria in cultures
of separately studied starter cultures and experiments repetitions.
DISCUSSION
Analysis of culture growth of selected lactic acid bacteria in medium with addition
of cholesterol solution and without it, provided in this study, proven lack of significant
influence of this addition on the dynamic of those bacteria growth. There is no informa-
tion in the available literature about any previous attempts to assess this influence. The
only exception is the study of Kimoto et al. [2002], who observed that the presence of
cholesterol stimulated, to some extent, the growth of cells of Lc. lactis subsp. lactis
biovar diacetylactis N7 strain. Kimoto et al. used GM17-THIO broth with 0.2% sodium
Technologia Alimentaria 6(1) 2007

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90
M. Ziarno ...
taurocholate as a medium for bacteria culture and bacterial growth was determined by
measuring the optical density of bacterial cells at 620 nm. This method of culture and
determination of bacterial growth differed from those used in this work, so it may be a
reason for divergent results.
As for the study regarding assessment of lactic acid bacteria ability to assimilate
cholesterol there is also no information in literature indicating that commercial starter
cultures of lactic acid bacteria have been studied. A large majority of studies referred to
in the literature regarded Lb. acidophilus strains of intestinal origin (isolated from hu-
mans or animals). Monocultures of Lb. acidophilus studied in this study, being dairy
starter cultures, were characterized by a high ability to assimilate cholesterol during
their growth and simultaneous reduction of its concentration in culture medium, com-
paring to the other studied strains. Value of this assimilation was, however, highly vari-
able depending on repetition.
Results of numerous other studies showed an existence of very significant differ-
ences in cholesterol assimilation ability between strains of individual lactic acid bacte-
ria, and frequently even between individual repetitions for the same strain [Gilliland et
al. 1985, Gilliland and Walker 1989, Buck and Gilliland 1994, Gopal et al. 1996, Tar-
anto et al. 1996, Pereira and Gibson 2002]. For example, Rasic et al. [1992] observed
ability of three Lb. acidophilus strains to assimilate cholesterol in MRS broth during
18 h culture in 37°C. The assimilation value for Lb. acidophilus strains was from 177 µg
to 225 µg in 1 cm3 of medium. Assimilation values obtained for Lb. acidophilus were
higher than for other species and strains of lactic acid bacteria studied by those scientists
(among others of bifidobacteria, yoghurt cultures of Str. salivarius subsp. thermophilus
and Lb. delbrueckii subsp. bulgaricus). For example, for Str. salivarius subsp. thermo-
philus strains the assimilation was only 59 and 69 µg of cholesterol in 1 cm3 of medium.
Walker and Gilliland [1993] found that 11 strains of Lb. acidophilus had the ability to
assimilate from 0 µmol of cholesterol in 1 cm3 to 38 µmol cholesterol in 1 cm3 of MRS
THIO medium with an addition of oxgall. Similar observations regarding differences in
assimilation between Lb. acidophilus strains were made by Gopal et al. [1996]. In their
study 6 strains of Lb. acidophilus assimilated from 15% to 55% of cholesterol from
MRS THIO medium with addition of oxgall.
A complete discussion of the results obtained in this study is hindered by the fact
that in the vast majority of published studies, MRS broth medium enriched with bile
salts, single (for example sodium taurocholate) or mixture of conjugated or deconju-
gated bile acid salts (for example oxgall) was used. Also various solutions and concen-
trations of cholesterol were used, as well as various time of culture. This assumption
was confirmed by Lin and Chen [2000], who studied cholesterol level reduction ability
of six strains of Lb. acidophilus during culture in three different media: one with addi-
tion of oxgall (mixture of conjugated and deconjugated bile salts), other with cholic acid
(source of deconjugated bile salts), and taurocholic acid (source of conjugated bile
salts). The studied strains assimilated from 20% to 57% cholesterol in presence of ox-
gall, from 43% to 71% cholesterol in presence of cholic acid, and 11-52% cholesterol in
presence of taurocholic acid.
Among lactic acid bacteria monocultures studied in this study, high ability of cho-
lesterol assimilation from MRS broth medium during 24 h incubation in 37ºC, was also
shown for probiotic monoculture of Bifidobacterium bifidum strain (Fig. 3). Active
assimilation of cholesterol by cultures of Bifidobacterium was also observed by Rasic et
al. [1992], who did not find, however, significant differences between the two studied
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Cholesterol assimilation by commercial yoghurt starter cultures
91
strains of B. bifidum (assimilation value of 174 µg of cholesterol in 1 cm3 and 138 µg of
cholesterol in 1 cm3, respectively). Tahri et al. [1996] observed completely different
ability to assimilate cholesterol by two strains of B. breve. For B. breve ATCC 15700
strain cholesterol assimilation value was 50%, and for B. breve ATCC 15698 strain –
9% only. The cited researchers found also that in seven studied strains of bifidobacteria
the measured value of assimilation was from 6% (in B. thermophilum ATCC 25525
strain) to 50% (in B. breve ATCC 15700 strain).
In this study the lowest ability of cholesterol assimilation was observed for bacteria
present in typical yoghurt starter cultures being a combination of cultures of Lb. del-
brueckii subsp. bulgaricus and Str. salivarius subsp. thermophilus. Those results are
thus slightly different from those obtained by Rasic et al. [1992]. Lactic acid bacteria
from commercial yoghurt culture studied by Rasic et al. bound 111 µg of cholesterol in
1 cm3 of MRS broth medium during 18 h incubation in 37ºC. One of Lb. delbrueckii
subsp. bulgaricus strain, studied by those researchers showed the highest assimilation
ability among all the cultures studied. It was 276 µg of cholesterol in 1 cm3 of MRS
broth. Whereas the studied cultures of Str. salivarius subsp. thermophilus cultures as-
similated less cholesterol comparing to Lb. delbrueckii subsp. bulgaricus cultures. So,
the researchers stated that cultures of thermophilic lactobacilli of Lb. delbrueckii subsp.
bulgaricus species much more actively assimilate cholesterol comparing to cultures of
thermophilic streptococci Str. salivarius subsp. thermophilus species. It must be noted,
however, that the ability of cholesterol assimilation may be influenced by numerous
factors, such as kind of medium, presence of bile salts, phase of bacterial growth, viabil-
ity and number of bacterial cells. Hosono and Tono-Oka [1995] and Usman and Hosono
[1999] suggest that the differences in the quantity of the assimilated cholesterol are
caused by chemical and structural properties of a peptidoglycan present in cellular walls
of those bacteria. According to them, this peptidoglycan possessing various amino acid
composition in various bacteria, is this component that makes cholesterol attachment
(assimilation) to cellular walls possible. However, no studies confirming this hypothesis
have been performed yet.
In this study interesting results were obtained in respect to yoghurt starter cultures
containing additional microflora Lb. acidophilus strains and Bifidobacterium species.
They confirm a high cholesterol assimilation ability of previously discussed Lb. aci-
dophilus strains and Bifidobacterium species. Basing on them it is possible to draw a
conclusion that those bacteria assimilate cholesterol equally well as monocultures and in
presence of other lactic acid bacteria. This is an interesting finding in view of the phe-
nomena of symbiosis or antagonistic growth between bacteria used for yoghurt and
bioyoghurt production.
Few literature data suggest ability of some lactic acid bacteria to reduce quantity of
cholesterol during production of fermented dairy beverages. This is also confirmed by
Polish studies carried out by Juskiewicz and Panfil-Kuncewicz [2003]. The researchers
showed that bacteria included in the tested thermophilic starter cultures possess ability
to reduce cholesterol contents in milk during its fermentation. Similarly to this study,
they observed that cholesterol assimilation level depended on the kind of the used cul-
ture and properties of the lactic acid bacteria present in each of those cultures, and also
– to a lesser extent – on the fat content in milk. The highest cholesterol assimilation
ability was shown by the bacteria present in one classic yoghurt starter culture, for
which assimilation value was 22.2% in yoghurt containing 4% fat, and 19.8% on yo-
Technologia Alimentaria 6(1) 2007

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M. Ziarno ...
ghurt containing 8% fat. Another yoghurt starter culture, with similar qualitative com-
position, showed cholesterol assimilation ability at the level of 11.3% and 15.4%, re-
spectively. In the cited study cholesterol was quite well assimilated also by Lb. aci-
dophilus strain (17.5% and 13.6%) and Bifidobacterium bifidum strain (12% and
13.5%). Lower cholesterol assimilation activity was shown by Str. salivarius subsp.
thermophilus strain (5.7% and 5.6%). Definitely the weakest cholesterol assimilation
was noted by the researchers in bacteria coming from a starter culture containing all the
discussed lactic acid bacteria – assimilation value was only 3.1% in yoghurt containing
4% fat and 3.2% in yoghurt containing 8% fat. Thus, a result of the cited study does not
overlap the results obtained in this study and concerning the cholesterol assimilation
from MRS broth medium. This may be explained by different dynamic of growth of the
used lactic acid bacteria in milk and culture medium, and also by other forms of choles-
terol present in milk and added to the MRS broth. In milk cholesterol is localized
mainly in phospholipids-protein membranes of fat globules, and cholesterol used in
experiments of this study was a solution of chemically pure substance.
This additional, potentially health-beneficial effect of thermophilic lactic acid bacte-
ria used in industrial practice constitutes an argument for continuation of this research.
It is not known, however, if this cholesterol binding to bacterial cells is stable enough to
resist conditions present in human intestinal tract, for example in high acidity, in pres-
ence of digestive enzymes of gastric juice or intestine juice. This would explain results
of clinical tests showing relationship between consumption of the fermented food prod-
ucts and reduction of cholesterol level in human and animal blood serum.
SUMMARY
1. Commercial yoghurt starter cultures are able to assimilate cholesterol from MRS
broth in absence of bile salts. The studied commercial starter cultures showed various
abilities to assimilate cholesterol from culture broth.
2. For five typical starter cultures, used for production of traditional yoghurt, the av-
erage cholesterol assimilation value was from almost 12% to over 21%. The cholesterol
assimilation by starter cultures used for bioyoghurt production was at the level of almost
18% to over 38%.
3. There is a significant difference in cholesterol assimilation ability between strains
of individual lactic acid bacteria, and also between individual repetitions for the same
strain.
4. The addition of cholesterol solution does not inhibit or stimulate the growth of
lactobacilli or streptococci included in commercial yoghurt starter cultures.
REFERENCES
Brashears M.M., Gilliland S.E., Buck L.M., 1998. Bile salt deconjugation and cholesterol re-
moval from media by Lactobacillus casei. J. Dairy Sci. 81, 2103-2110.
Buck L.M., Gilliland S.E., 1994. Comparisons of freshly isolated strains of Lactobacillus aci-
dophilus of human intestinal origin for ability to assimilate cholesterol during growth. J.
Dairy Sci. 77, 2925-2933.
Acta Sci. Pol.

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