European Journal of Clinical Nutrition (2000) 54, 288±297
ß 2000 Macmillan Publishers Ltd All rights reserved 0954±3007/00 $15.00
www.nature.com/ejcn
Effect of 8 week intake of probiotic milk products on risk factors
for cardiovascular diseases
L Agerholm-Larsen*, A Raben, N Haulrik, AS Hansen, M Manders and A Astrup
Research Department of Human Nutrition, Centre for Food Research, The Royal Veterinary and Agricultural University, DK-1958
Frederiksberg C, Denmark
Objective: To investigate the effect of a probiotic milk product containing the culture CAUSIDO1 and of two
alternative products on risk factors for cardiovascular disease in overweight and obese subjects.
Design: An 8 week randomized, double-blind, placebo- and compliance-controlled, parallel study.
Subjects: Seventy healthy, weight-stable, overweight and obese (25.0 < BMI < 37.5 kg=m2) males (n ˆ 20) and
females (n ˆ 50), 18 ± 55 y old, were randomly assigned into ®ve groups.
Intervention: Four groups consumed 450 ml fermented milk products (yoghurt) daily. Group 1: a yoghurt
fermented with two strains of Streptococcus thermophilus and two strains of Lactobacillus acidophilus (StLa).
Group 2: a placebo yoghurt fermented with delta-acid-lactone (PY). Group 3: a yoghurt fermented with two
strains of Streptococcus thermophilus and one strain of Lactobacillus rhamnosus (StLr). Group 4: a yoghurt
fermented with one strain of Enterococcus faecium and two strains of Streptococcus thermophilus (CAUSIDO1
culture), GAIO1 (G). The dietary composition of the yoghurt was otherwise similar. The ®fth group was given
two placebo pills (PP) daily.
Results: When comparing all ®ve treatment groups, unadjusted for changes in body weight, no statistical effects
were observed in week 8 in the G-group on low density lipoproteins (LDL)-cholesterol (P ˆ 0.29). After
adjustment for small changes in body weight, LDL-cholesterol decreased by 8.4% (0.26 0.10 mmol=l;
P < 0.05) and ®brinogen increased (0.74 0.32 mmol=l; P < 0.05) after 8 weeks in the G-group. This was
signi®cantly different from the group consuming chemically fermented yoghurt and the group consuming
placebo pills (P < 0.05). After 8 weeks, systolic blood pressure was signi®cantly more reduced in the StLa and Ggroup compared to StLr. No other differences were found.
Conclusion: The CAUSIDO1 culture reduced LDL-cholesterol and increased ®brinogen in the overweight
subjects at a 450 ml consumption daily for 8 weeks. The effect on LDL-cholesterol con®rms previous studies. An
immunostimulation by one of the strains in the product might explain the effect on ®brinogen in the G-group.
Sponsorship: MD Foods A=S, Denmark.
Descriptors: gut bacteria cultures; cholesterol; body weight; ®brinolysis; coagulation; compliance
European Journal of Clinical Nutrition (2000) 54, 288±297
Introduction
It is well known that cardiovascular disease (CVD) is a
multifactorial disease with a high mortality rate and that it
is one of the most important causes of death in Denmark
and in several other Western countries. One of the major
risk factors for CVD is abnormal levels of blood lipids. In
particular, a high level of low density lipoprotein (LDL)cholesterol in the blood increases the risk of developing
CVD (Grundy, 1997).
Recently, investigations have been performed concerning the effects of the fermented milk product GAIO1,
*Correspondence: L Agerholm-Larsen, Research Department of Human
Nutrition, Centre for Food Research, The Royal Veterinary and
Agricultural University, DK-1958 Frederiksberg C, Denmark.
E-mail: [email protected]
Guarantor: Arne Astrup.
Contributors: A Raben and A Astrup initiated the project, which also was a
part of a bachelor-project of N Haulrik and AS Hansen. M Manders was
responsible for the compliance pilot-study and the compliance
measurements as part of her student exchange programme (ERASMUS) at
the department. All the authors were involved in the carrying out the
experimental work. N Haulrik, AS Hansen and L Agerholm-Larsen made
the statistical analysis. L Agerholm-Larsen wrote the article in
collaboration with A Raben and A Astrup.
Received 26 June 1999; revised 28 September 1999; accepted 9 October
1999
containing the bacterial culture CAUSIDO1, on risk factors for CVD. One study observed that 200 ml GAIO1
consumed daily for 6 weeks reduced LDL-cholesterol by
10% in a homogeneous group of normal-weight, middleaged males (Agerbñk et al, 1995). A subsequent study
investigated whether the possible cholesterol-lowering
effect of GAIO1 was sustained for more than 6 weeks
(Richelsen et al, 1996). In this 6 month long-term study,
GAIO1 showed a rapid reduction in LDL-cholesterol level
by about 8% after 1 month, but after the entire intervention
the reduction in LDL-cholesterol was similar to reduction
induced by the placebo product. This may, however, be due
to decreasing compliance during the late part of the study
period or to lack of statistical power to detect a 4 ± 5%
reduction in blood cholesterol. Another very recent study,
based on the above studies on GAIO1, evaluated GAIO1's
effects on primary hypercholesterolaemia (Bertolami et al,
1999). It was different compared with the Danish studies, in
that it used an 8 week cross-over design and different kinds
of subjects. Bertolami et al (1999) used non-obese, moderately hypercholesterolaemic women and men who had
not earlier shown an improvement in LDL-cholesterol level
by dietetic modi®cations alone. The results showed that
GAIO1 was able to reduce LDL-cholesterol by about 6%
in these hypercholesterolaemic subjects compared with a
Probiotic milk products and cardiovascular risk
L Agerholm-Larsen et al
placebo product. The impact of GAIO1 on cholesterol
levels in obese subjects has, however, not yet been investigated.
The present investigation had the following aims: (1) to
investigate the effect of GAIO1 in obese subjects with a
strict dietary compliance. This was obtained by controlling
the intake of the product and by increasing the daily
dosage. (2) To examine whether the bene®cial effects on
risk factors previously observed in the GAIO1 group
(Richelsen et al, 1996) can be attributed to the macro- or
micronutrients shared by GAIO1 and the control product.
This was done by having two control groups, one receiving
a placebo tablet and another receiving a chemically acidi®ed yoghurt. (3) To examine whether any bene®cial effect
on CVD risk factors is speci®c for GAIO1 or may be
achieved by other bacterial culture strains with favourable
in vitro properties.
Methods
Experimental design
The study was designed as a randomized, double-blind,
placebo- and compliance-controlled, parallel study performed for 8 weeks. The study was designed as a ®vearmed parallel study in which GAIO1 was compared with
two other fermented milk products, a chemically acidi®ed
milk product and inert placebo pills. It had been decided in
advance that if no signi®cant changes in lipoproteins were
observed after 4 weeks of yoghurt consumption, the intervention period should stop after 8 weeks. Blood results
from week 4 did not show any signi®cant differences
between groups. Therefore, the study stopped after 8
weeks. The present study was designed to offer an
increased amount of yoghurt (450 ml daily) to obtain a
greater effect on LDL-cholesterol. Furthermore, subjects
were consuming at least 900 ml test-yoghurt weekly at the
department (three visits), to increase compliance. Dietary
compliance tests were also performed at home once every
second week. Overweight subjects were used, since they
usually have higher levels of blood lipids and therefore are
at increased risk of CVD compared with normal-weight
subjects.
An information meeting was held one month or earlier
before the intervention. The subjects were told about the
inclusion criteria verbally and in writing. They were
instructed not to change their habitual diet, level of physical
exercise, tobacco and alcohol habits or their body weight
during the intervention period. The participants were also
instructed not to consume any kinds of fermented milk
products 1 month before and during the 8 week intervention
except the 450 ml test yoghurt daily. The subjects attended
the department 3 days a week (mornings or afternoons) to
consume 300 ml yoghurt or one placebo tablet and to
collect products for consumption at home. The yoghurt
was presented in identical plastic cups containing 150 ml.
Each cup had a colour band (red, green, yellow or blue) for
the different intervention groups. It was not known for the
subjects that one product was the well-known yoghurt
product GAIO1. The subjects were instructed to replace
some food items, especially similar milk products, with the
daily given yoghurt. The placebo pill group was following
the same protocol as the yoghurt groups except consuming
an inert placebo pill instead of yoghurt.
In week 0, 4 and 8 the subjects were weighted, body
composition and blood pressure were measured and blood
samples were taken. A dietician also instructed them
verbally and in writing how to keep a 7-day weighed
dietary record in week 0 and 8. Each day during the
intervention the subjects ®lled in a short diary about their
well-being, physical activity, defecation pattern, any use of
medicine and appetite sensations. Compliance to the
yoghurt consumption at home was measured once every
second week during the intervention (weeks 2, 4, 6 and 8).
After the intervention period the subjects were offered a
slimming course as a reward for their participation in the
study. All subjects also received 1000 DKK after completion of the intervention period. The Municipal Ethical
Committee of Copenhagen and Frederiksberg approved
the study as according to the Helsinki-II declaration. All
subjects gave written consent after the experimental procedure had been carefully explained to them in writing and
at an information meeting.
289
Subjects
Subjects were recruited after advertisement in national and
local newspapers and by sending letters to subjects on a
waiting-list for weight loss at the department. Inclusion
criteria were: healthy (no diabetes, kidney or liver disease);
normal blood pressure; overweight (body mass index
(BMI): 25.0 < BMI < 37.5 kg=m2); males and females (premenopausal); 18 ± 55 y old; no medications (except birth
control pills); not pregnant or breast-feeding; no special
diets (eg vegetarian); normal alcohol habits and not elite
athletes (Table 1). The subjects were screened anthropometrically, by blood samples and by urine sticks before
being included in the study. Our ambition for the study was
to recruit 100 participants. About 800 information letters
were sent to interested subjects. After this, 80 subjects were
still interested, quali®ed and were appointed to the study.
However, before the intervention started, seven subjects
had second thoughts and dropped out. We carefully divided
the remaining 73 subjects into ®ve groups matched by
gender, age, BMI, fat-free mass (FFM), fat mass (FM), total
high density lipoprotein (HDL)- and LDL-cholesterol. The
matching was performed blinded by a person with no
contact with the subjects. About 15 subjects were assigned
to each yoghurt group and 10 to the placebo pill group.
Four of the groups consumed 450 ml fermented milk
product (yoghurt) daily. One group consumed the test
product GAIO1 (G), two groups consumed two different
and new kinds of fermented milk products (StLa and StLr)
and one group consumed a chemically fermented milk
product (placebo yoghurt) (PY). The ®fth group was
given two placebo tablets daily (PP). The placebo tablets
each contained 500 mg calcium lactate corresponding to
65 mg calcium (Nycomed1).
During the intervention period three female subjects
dropped out. One subject got pregnant (group PY) and
two experienced nausea and constipation after a few weeks
of yoghurt consumption (group PY and StLr). Thus, 70
subjects completed all 8 weeks of the intervention. Their
characteristics are given in Table 1).
Diets
The Danish dairy corporation MD Foods (Aarhus, Denmark) produced the four fermented milk products. The test
yoghurt GAIO1 (G) was fermented using the original
Ukrainian bacterial culture CAUSIDO1. This culture contained one strain of Enterococcus faecium (human species)
and two strains of Streptococcus thermophilus. The colonyEuropean Journal of Clinical Nutrition
Probiotic milk products and cardiovascular risk
L Agerholm-Larsen et al
290
Table 1 Subject characteristics before intervention (n ˆ 70)
n
Sex (f=m)
Age (y)
Body weight (kg)
Height (cm)
Body mass index (kg=m2)
Fat mass (kg)
Fat-free mass (kg)
Blood pressure (systolic) (mmHg)
Blood pressure (diastolic) (mmHg)
Sagittal heights (cm)
Waist ± hip ratio (cm)
Total cholesterol (mmol=l)
Triacylglycerol (mmol=l)
HDL-cholesterol (mmol=l)
LDL-cholesterol (mmol=l)
Fibrinogen (mmol=l)
tPA activity (IU=ml)
FVIIc (%)
StLa
PY
StLr
G
PP
ANOVA P-value for
group effect
16
12=4
38.6 2.1
85.9 2.7
169 2.1
30.0 0.7
32.2 1.7
53.7 2.0
121.4 2.8
78.9 2.3
21.9 0.7
0.88 0.03
5.13 0.22
1.43 0.14
1.29 0.09
3.19 0.20
10.4 0.4
0.36 0.03
1.2 0.04
14
9=5
39.4 2.1
87.9 4.1
171 2.6
30.0 0.9
32.6 2.0
55.3 2.8
116.5 3.8
76.4 2.9
22.0 0.8
0.92 0.04
5.32 0.32
2.00 0.32
1.30 0.09
3.14 0.22
10.5 0.4
0.45 0.06
1.3 0.1
14
10=4
37.9 2.4
90.5 5.2
172 3.4
30.2 0.7
33.6 2.0
56.9 3.6
121.6 5.2
75.9 3.7
22.3 0.8
0.88 0.03
5.16 0.28
1.56 0.24
1.36 0.09
3.09 0.22
9.9 0.3a
0.36 0.05a
1.2 0.04a
16
12=4
37.8 2.0
88.9 4.1
171 2.9
30.1 0.6
32.6 1.3
56.3 3.2
131.9 6.9
83.0 5.2
22.0 0.7
0.87 0.02
5.06 0.22
1.65 0.20
1.30 0.10
3.01 0.20
10.4 0.3b
0.53 0.08b
1.1 0.04b
10
7=3
38.3 3.2
85.5 3.7
169 2.5
29.9 1.1
30.6 2.1
49.5 6.09
122.0 4.2
80.3 3.4
21.8 0.6
0.86 0.03
5.01 0.24
2.13 0.53
1.33 0.18
2.81 0.24d
10.2 0.3c
0.31 0.05c
1.4 0.1c
Ð
0.99
0.91
0.94
1.00
0.95
0.65
0.25
0.64
0.99
0.77
0.90
0.37
0.99
0.83
0.98
0.47
0.46
Data are means s.e.m. n ˆ 70 subjects. StLa, Streptococcus thermophilus ‡ Lactobacillus acidophilus; PY, placebo yoghurt; StLr, Streptococcus
thermophilus ‡ Lactobacillus rhamnosus; G, GAIO1; PP, placebo pill; f=m, females=males; HDL-cholesterol, high density lipoprotein cholesterol;
LDL-cholesterol, low density lipoprotein cholesterol.
a
n ˆ 13; bn ˆ 15; cn ˆ 8; dPP, n ˆ 9.
forming units (CFU) of the fresh product were about
66107=ml for Enterococcus faecium and 16109=ml
for Streptococcus thermophilus. As alternative test
yoghurts, two new compositions of bacteria cultures were
used. The choice of bacteria strains was based on screening
of a number of strains' in vitro properties to affect cholesterol in a bene®cial way. Several in vitro and animal studies
have shown that removal=assimilation of cholesterol is
related to the ability of a culture strain to deconjugate
bile salts, such as Enterococcus faecium (Gilliland &
Speck, 1977; Gilliland et al, 1985; Taranto et al, 1996).
However, tolerance to bile is essential to secure the strain's
survival on the way through the gastrointestinal system. A
pH lower than 5.5 deconjugates bile acids and cholesterol
co-precipitate which are both excreted in faeces (Klaver &
Van der Meer, 1993), while a neutral or alkaline pH, as in
the human small intestine, makes it more dif®cult for some
strains to survive in vivo. However, it is still not clear
which strains possess the same ability in vitro as in vivo in
humans. One of the alternative yoghurt products (StLr) had
a strain composition that, according to an in vitro study by
MD Foods, had similar abilities to assimilate cholesterol as
the CAUSIDO1 culture in GAIO1. The other alternative
(StLa) was expected to have a good ability to both conjugate bile salts and assimilate cholesterol. Both test
yoghurts had almost similar taste and consistency as the
GAIO1 product. The test yoghurt StLa was fermented with
two strains of Streptococcus thermophilus (CFU ˆ
106107=ml) and two strains of Lactobacillus acidophilus (CFU ˆ 26107=ml). The second test yoghurt StLr
contained two strains of Streptococcus thermophilus
(CFU ˆ 86108=ml) and one strain of Lactobacillus
rhamnosus (CFU ˆ 26108=ml) (StLr). The placebo
milk product was of identical composition as the other
milk products, but chemically fermented with an organic
acid (delta-acid-lactone) instead of a living bacterial culture
(PY). 100 g of all products contained 224 kJ (54 kcal),
5.4 g protein, 3.3 g carbohydrate and 1.7 g fats (1%
milk fat and 0.7% rapeseed oil). The cholesterol
content was 3 ± 4 mg=100 g yoghurt. The products were
European Journal of Clinical Nutrition
manufactured and sent to the department about every
second week and the yoghurt was kept in a refrigerator at
max. 5 C.
Anthropometric measurements
Body weight, body composition and blood pressure were
measured in weeks 0, 4 and 8 in the morning after 10 h of
fasting. For body weight the same digital scale was used
each time (Lindeltronic model 8000 C, Frederiksberg,
Denmark). Body composition was subsequently estimated
by electrical bioimpedance using an Animeter (HTS-Engineering Inc., Odense, Denmark). Fat mass (FM) and fatfree mass (FFM) were calculated using equations by
Heitmann (1990). For waist ± hip ratio (WHR) and sagittal
height (visceral fatness) the same tape measure and sagittal
measure were used, respectively. Blood pressure was measured using an automatically in¯ating cuff (Digital Blood
Pressure Meter, model UA-746, A&D Company Ltd,
Tokyo, Japan).
Laboratory sampling and analyses
Blood samples were drawn in the morning after 10 h of
fasting in weeks 0, 4 and 8 of the intervention. The subjects
rested on a bed in the supine position for 10 min before the
blood sample was taken. The subjects were not allowed to
use any medications 2 days before the blood sample and
they also had to abstain from tobacco smoking on the
sampling day. Blood was sampled without stasis from the
antecubital vein in the arm. The blood was analysed for
triacylglycerol, total cholesterol, HDL=LDL-cholesterol,
tissue-type plasminogen activator (tPA activity), factor
VII coagulant activity (FVIIc) (human tissue-factorinduced), ®brinogen, and C-reactive protein (CRP). The
results of triacylglycerol, total cholesterol, HDL=LDLcholesterol and CRP from week 0, 4 and 8 and tPA,
FVIIc and ®brinogen from week 0 and 8 are presented in
this publication.
The blood for triacylglycerol, total cholesterol and
HDL=LDL-cholesterol analyses was collected in tubes
without anticoagulants. Samples for triacylglycerol, total
Probiotic milk products and cardiovascular risk
L Agerholm-Larsen et al
cholesterol and HDL=LDL-cholesterol were centrifuged for
15 min at 3000 g at 20 C. The samples were stored at
7 20 C until further analyses. Serum was analysed by
standard enzymatic methods. For total cholesterol and
triacylglycerol Monotest Cholesterol High Performance
(CHOD-PAP) and Test-Combination Triacylglycerol
(GPO-PAP) methods from Boehringer Mannheim GmbHDiagnostica (Germany) were used, respectively (Seidel et
al, 1983; Kattermann et al, 1984; Wahlefeld, 1974). HDLcholesterol was measured after sedimentation of apo-lipoprotein-B-containing lipoproteins with magnesium-phosphorus-wolfram acid (the precipitant method; Burstein et
al, 1970; Lopes-Virella et al, 1977) also from Boehringer,
Mannheim. The plasma was measured on a Cobas Mira S
(Boehringer Mannheim GmbH, Germany). The content of
LDL-cholesterol was calculated from the analysed values
of total cholesterol, HDL-cholesterol and triacylglycerol:
LDL-cholesterol (mmol=l) ˆ total cholesterol (mmol=l) 7
(triacylglycerol (mmol=l)=2.2) 7 HDL-cholesterol (mmol=l)
(modi®ed after Friedewald et al, 1972).
Blood for tPA analyses was collected in speci®c prechilled tubes containing citrate (Biopool StabilyteTM, Biopool, Sweden). Within 2 h the plasma was centrifuged at
3000 g at 4 C for 15 min and stored at 7 80 C until further
analysis. For determination of tPA activity a biofunctional
immunosorbent assay (BIA) was used (Chromolize tPATM,
Biopool, Sweden; RaÊnby et al, 1994). Blood for FVIIc and
®brinogen analyses was collected in tubes containing
citrate. Within 1 h the blood was centrifuged at 3000 g at
20 C in 15 min and plasma stored at 7 80 C until further
analyses. FVIIc was analysed using a one-stage clotting
assay using human FVIIc de®cient plasma (Biopool,
Sweden) and a human placenta tissue thromboplastin
factor (TF) (Thromborel S, Behringwerke AG, Germany)
(more details described in Larsen et al, 1997). For determination of ®brinogen concentration a one-stage clotting
assay with bovine thrombin was used, a functional method
modi®ed after Clauss (1957) (described in Jespersen &
Sidelmann, 1982). FVIIc and ®brinogen were analysed on
an ACLTM 100 (Automated Coagulation Laboratory,
Instrumentation Laboratory, Italy). For determination of
CRP the immuno-turbidimetry principle was used with
antibodies (DAKO1, Denmark) containing rabbit antihuman CRP. CRP was analysed on a Cobas Fara II (Roche1,
Schweiz). The reference interval was 0 ± 10 mg=l with 10%
precision.
The subjects were screened for normal levels of
HDL=LDL-cholesterol and blood glucose (diabetes)
before inclusion in the study. Screening urine with urine
sticks determined glucose and ketone bodies (BMTest-9, In Vitro Diagnossticum, Boehringer, Mannheim,
Germany).
Compliance
Dietary compliance was measured at home once every
second week (weeks 2, 4, 6 and 8). The subjects were
told that the test was done to investigate how the yoghurt
was digested and absorbed from the intestine. Subjects
collected exhalation air in special breath sampling bags
(Wagener Analyzen Technik, Bremen, Germany) before
and 1 h after consuming one 13C-enriched yoghurt. The
dose and the right time for collecting the samples were
determined in a pilot study before the intervention. For
measuring compliance, 13C-acetate (Wagener Analyzen
Technik, Bremen, Germany) was dissolved in water
(100 mg acetate in 1 ml water) and injected with an insulin
needle to the plastic cup containing 150 ml yoghurt. The
13
C-acetate was added about 2 days before consumption.
The cup was shaken to mix the 13C-acetate with the
yoghurt. For each subject the enriched yoghurt was then
marked with a date on which it should be consumed at
home. The subjects did not have to be fasting, but during
the sampling they were not allowed to consume any food or
to engage in heavy physical exercise. The sampling bags
were analysed when possible after maximum 1 ± 2 days
after the subjects had delivered them at the department. The
bags were analysed for 13CO2=12CO2 ratio by the nondispersive infrared spectroscopy method (NDIRS) (Wagener Analyzen Technik, Bremen, Germany). This method
measures delta over baseline (DOB) for 13CO2=12CO2 ratio.
From the pilot study it was decided that a DOB level of 6%
or more was the limit to show that the enriched yoghurt had
been consumed. The placebo tablet group was asked to do
the compliance-test without any enriched yoghurt to ensure
the same protocol for all subjects.
291
Statistical analyses
All results are given as means standard error of mean
(s.e.m.). Data are analysed and presented as changes from
initial fasting levels (week 0 ˆ baseline). For matching the
groups, Super Calc version 5.0 (Computer Associates
International Inc.) was used. Changes from baseline
within a group were tested by Statgraphics Software version 4.2 (Graphic Software Systems Inc., Rockville, MD).
Differences between groups were tested by parametric
analysis of variance (ANOVA) using the GLM procedure
in SAS version 6.12 (SAS institute Inc., Cary, NC). Data
were also analysed with changes in body weight and fat
mass (FM) as covariates. Correlation analyses were performed by Statgraphics Software version 4.2. The signi®cance level was set at P < 0.05.
Results
Body weight (BW) and composition
After 4 weeks intervention a small increase in BW was
found in all ®ve groups, being signi®cant in the groups
consuming PY (0.73 0.23 kg), G (0.58 0.22 kg) and PP
(1.35 0.14 kg) (P < 0.05). No signi®cant differences were
found among the ®ve groups (P ˆ 0.14).
In week 8 one female subject from the G-group was ill a
couple of days before the anthropometrical measurement.
Since the subject therefore lost > 2 kg, she was excluded
from the statistical analyses for BW, FM and BMI (Ggroup: n ˆ 15). BW increased in all ®ve groups (P < 0.05),
except in the PY-group (P ˆ 0.06). There were no differences between the groups (P ˆ 0.81; Table 2). After 8
weeks FM also increased in the groups consuming StLa,
G and PP (P < 0.05), but no signi®cant group differences
were found (P ˆ 0.81; Table 2).
After week 8 the StLa and StLr group had an increased
WHR compared to week 0 (P < 0.05), however, no group
differences were found (P ˆ 0.20; data not shown). After 8
weeks intervention no differences were found in sagittal
height (data not shown).
Blood lipids
After 4 weeks intervention no signi®cant alterations or
group differences were found in any of the primary lipid
effect parameters (total cholesterol, HDL- or LDL-cholesEuropean Journal of Clinical Nutrition
Probiotic milk products and cardiovascular risk
L Agerholm-Larsen et al
292
Table 2 Changes in anthropometric parameters after 8 weeks of intervention
n
DBody weight (kg)
DFat mass (kg)
DBP (systolic) (mmHg)
DBP (diastolic) (mmHg)
StLa
PY
StLr
Ga
PP
ANOVA P-value
for group effect
16
0.99 0.26**
0.98 0.31**
7 4.4 1.8*A
7 3.4 1.5*
14
1.06 0.53§
0.48 0.52
7 2.2 1.9
7 1.5 1.3
14
0.73 0.31*
0.60 0.30§
2.6 3.1B
0.8 2.0
16
0.59 0.24*
0.75 0.29*
7 8.0 2.3**A
7 4.0 2.4
10
1.01 0.17***
0.98 0.17***
7 4.4 2.2
7 6.1 2.5*
0.81
0.81
0.02
0.19
Data are means s.e.m. n ˆ 70 subjects.
A female subject was excluded from the G group (n ˆ 15) because of illness prior to the measurement.
StLa, Streptococcus thermophilus ‡ Lactobacillus acidophilus; PY, placebo yoghurt; StLr, Streptococcus thermophilus ‡ Lactobacillus rhamnosus;
G, GAIO1; PP, placebo pill; D ˆ changes from baseline (week 8 7 week 0); BP ˆ blood pressure.
*P < 0.05; **P < 0.01; ***P < 0.001; §, P ˆ 0.06 compared to baseline (0.0).
Different letters (A, B): groups differ (P < 0.05). A negative value shows a reduction from baseline value (week 8 7 week 0).
a
Table 3 Lipoproteins, ®brinolysis and coagulation parameters after 4 and 8 weeks of intervention (changes from baseline)
ANOVA all groups
n
DTotal cholesterol (mmol=l)
DHDL-cholesterol (mmol=l)
DLDL-cholesterol (mmol=l)
DTriacylglycerol (mmol=l)
DFibrinogen (mmol=l)
DtPA activity (IU=ml)
DFVIIc (%)
Week
StLa
PY
StLr
G
PP
P-value
adj.DBW
4
8
4
8
4
8
4
8
8
8
8
16
0.05 0.13
0.05 0.11
7 0.01 0.03
7 0.02 0.03
7 0.10 0.11
0.12 0.01
0.35 0.14
7 0.01 0.09
0.04 0.26e
0.02 0.04e
7 3 0.02e
14
7 0.15 0.11
0.06 0.17
7 0.05 0.09
7 0.06 0.09
7 0.04 0.12
0.11 0.11
7 0.15 0.19
7 0.01 0.28
7 0.40 0.27
7 0.01 0.08
7 12 0.06
14
7 0.02 0.14
7 0.03 0.20
0.01 0.05
7 0.04 0.05
7 0.14 0.14a
7 0.01 0.19
0.42 0.27
0.04 0.24
0.08 0.43f
7 0.04 0.06f
4 0.08f
16
0.02 0.13
7 0.26 0.15
0.03 0.05
7 0.04 0.04
7 0.02 0.12b
7 0.20 0.10*
0.10 0.20
7 0.03 0.15
0.74 0.32g*
7 0.09 0.11g
7 0.3 0.04g
10
0.01 0.12
7 0.04 0.11
0.03 0;04
7 0.04 0.04
0.08 0.14c
0.16 0.17d
7 0.01 0.47
7 0.31 0.36
0.19 0.25
7 0.01 0.05
7 6 0.04
0.84
0.54
0.82
0.98
0.84
0.29
0.44
0.87
0.16
0.85
0.23
0.71
0.26
0.80
0.98
0.54
0.09
0.44
0.86
0.08
0.47
0.27
Data are means s.e.m.
StLa, Streptococcus thermophilus ‡ Lactobacillus acidophilus; PY, placebo yoghurt; StLr, Streptococcus thermophilus ‡ Lactobacillus rhamnosus; G,
GAIO1; PP, placebo pill; ANOVA: P values for group effects. D ˆ changes from baseline (week 8 7 week 0); adj.DBW, adjusted for changes in body
weight. HDL, high density lipoprotein; LDL, low density lipoprotein; tPA, ®brinolytic activity.
*P < 0.05 (different from baseline for G vs PP=PY, adjusted for changes in body weight; see Table 4).
a
n ˆ 12; bn ˆ 15; cn ˆ 8; dn ˆ 9; en ˆ 15; fn ˆ 12; gn ˆ 15.
terol, triacylglycerol) (Table 3). Based on these results, the
intervention was stopped after 8 weeks.
In week 8 there was a missing value from a female in the
PP group, because of a very high level of triacylglycerol
(hypertriglyceridaemia). According to the analysis (Friedewald, 1972), triacylglycerol values above 4.58 mmol=l
makes it impossible to calculate correct LDL-cholesterol
values. After 8 weeks we observed a signi®cant decrease
from baseline in LDL-cholesterol in the G-group, corresponding to 0.26 0.10 mmol=l (8.4%) (P < 0.05; Figure
1). However, there were no signi®cant differences in LDLcholesterol between groups (Table 3). After adjustment for
changes in BW there was still no signi®cant group difference when all ®ve groups were compared (P ˆ 0.09; Figure
1). However, when comparing the yoghurt group G with
the placebo groups PY and PP and thereby excluding the
groups StLa and StLr, the group effect after adjustment for
BW changes was signi®cant (P ˆ 0.03; Table 4). If comparing the G-group with the other two yoghurt groups (StLa
and StLr) and thereby excluding the placebo groups, the
group effect without (P ˆ 0.22) and with (P ˆ 0.06) adjustment for BW changes was not signi®cant (Table 4).
Total cholesterol and HDL-cholesterol in week 8
showed no signi®cant changes from baseline or group
differences without or with adjustments for changes in
European Journal of Clinical Nutrition
BW (Table 3). Triacylglcyerol was also similar in all ®ve
groups after 8 weeks of yoghurt consumption (Table 3).
Adjustments for changes in fat mass did not result in any
signi®cant differences among the ®ve groups (data not
shown).
Fibrinolysis and coagulation parameters
After 8 weeks intervention ®brinogen increased in the
G-group from the baseline value (week 0)
(0.74 0.32 mmol=l; P < 0.05; Figure 2). No group differences were found (P ˆ 0.16), not even after adjustment for
changes in BW (P ˆ 0.08; Table 3). When comparing the
G-group with PY and PP, a group effect was found
(P ˆ 0.02) with the G-group differing from the PY group
(P < 0.01; Table 4). We observed the same after adjustment
for changes in BW (Figure 2). When comparing the Ggroup with the two yoghurt groups (StLa and StLr) by
excluding the placebo groups, the group effect without and
with adjustment for changes in BW was not signi®cant for
®brinogen (Table 4).
No changes from baseline and no group differences were
seen for tPA activity and FVIIc (Table 3). When comparing
G with the yoghurt groups or placebo groups, respectively,
no differences were found either (Table 4).
Probiotic milk products and cardiovascular risk
L Agerholm-Larsen et al
293
Figure 1 Changes in LDL-cholesterol, week 8 7 week 0 adjusted for changes in BW. Data are means. StLa, Streptococcus thermophilus ‡ Lactobacillus
acidophilus; PY, placebo yoghurt; StLr, Streptococcus thermophilus ‡ Lactobacillus rhamnosus; G, GAIO1; PP, placebo pill. Group effect: P ˆ 0.09.
*P < 0.05 compared to 0.0.
Table 4 Comparison between G and placebo groups (PY and PP) and G and yoghurt groups (StLa and StLr) for
lipoproteins, ®brinolysis and coagulation parameters after 8 weeks of intervention (changes from baseline)
ANOVA G vs PY=PP
DTotal cholesterol (mmol=l)
DHDL-cholesterol (mmol=l)
DLDL-cholesterol (mmol=l)
DTriacylglycerol (mmol=l)
DFibrinogen (mmol=l)
DtPA activity (IU=ml)
DFVIIc (%)
ANOVA G vs StLa=StLr
Week
P-value
adj. DBW
P-value
adj. DBW
8
8
8
8
8
8
8
0.29
0.94
0.07
0.69
0.02
0.75
0.17
0.16
0.96
0.03
0.69
0.02
0.30
0.17
0.32
0.91
0.22
0.96
0.27
0.60
0.65
0.14
0.73
0.06
0.98
0.19
0.32
0.69
ANOVA, P values for group effects. G, GAIO1; PY, placebo yoghurt; PP, placebo pill; StLa, Streptococcus
thermophilus ‡ Lactobacillus acidophilus; StLr, Streptococcus thermophilus ‡ Lactobacillus rhamnosus; D ˆ changes
from baseline (week 8 7 week 0); adj.DBW, adjusted for changes in body weight. HDL, high density lipoprotein;
LDL, low density lipoprotein; tPA, ®brinolytic activity. G vs PP=PY, GAIO1 compared with the two placebo
products. G vs StLa=StLr, GAIO1 compared with the two yoghurt groups.
C-reactive protein
In weeks 0, 4 and 8, we found 12 values of CRP higher than
5 mmol=l, spread over all ®ve groups (data not shown). The
most extreme value was 118 mmol=l which belonged to the
excluded female from the G-group, who was ill before
blood sample day in week 8.
Blood pressure
After 8 weeks analyses of changes in systolic blood
pressure showed signi®cant differences between the
groups (P < 0.05). Thus, StLa and G were lower than
StLr (P < 0.05; Table 2). Adjustment for changes in BW
showed a similar group difference. The diastolic blood
pressure decreased signi®cantly in the StLa and PP-group
(P < 0.05), but there were no group differences (P ˆ 0.19;
Table 2), not even after adjustment for changes in BW.
Correlation analyses
Correlations were found after week 8 between changes in
total cholesterol and BW (r ˆ 7 0.33, P < 0.05), between
changes in HDL-cholesterol and BW (r ˆ 7 0.25,
P < 0.05) and between changes in LDL-cholesterol and
BW (r ˆ 7 0.30, P < 0.05). However, no signi®cant relationship was found between changes in BW and ®brinogen
(r ˆ 7 0.16, P ˆ 0.19) or between changes in LDL-cholesterol and ®brinogen (r ˆ 0.12, P ˆ 0.33). Changes in
total cholesterol and HDL-cholesterol (r ˆ 0.22, P ˆ 0.06)
and total cholesterol and ®brinogen (r ˆ 0.21, P ˆ 0.09) did
not show a signi®cant relationship.
European Journal of Clinical Nutrition
Probiotic milk products and cardiovascular risk
L Agerholm-Larsen et al
294
Figure 2 Changes in ®brinogen, week 8 7 week 0 adjusted for changes in BW. Data are means. StLa, Streptococcus thermophilus ‡ Lactobacillus
acidophilus; PY, placebo yoghurt; StLr, Streptococcus thermophilus ‡ Lactobacillus rhamnosus; G, GAIO1; PP, placebo pill. Group effect: P ˆ 0.08.
*P < 0.05 compared to 0.0.
Table 5 Energy and macronutrient content of the habitual diet in weeks 0 and 8
Energy (total) (kJ=day)
Carbohydrates (E%)
Sugar (total) (E%)
Sugar added (E%)
Fat (total) (E%)
Saturated fat (E%)
Monounsaturated fat (E%)
Polyunsaturated fat (E%)
Protein (E%)
Alcohol (E%)
Cholesterol (mg=day)
Fibre (g=day)
Weight of food (g=day)
Week
StLa
(n ˆ 14)
PY
(n ˆ 12)
StLr
(n ˆ 12)
G
(n ˆ 15)
PP
(n ˆ 8)
ANOVAa
P-value
ANOVAb
P-value
0
8
0
8
0
8
0
8
0
8
0
8
0
8
0
8
0
8
0
8
0
8
0
8
0
8
10307.9 718.6
9845.8 625.1
50.0 1.8
47.2 1.4
21.3 1.7
21.1 1.7
11.5 1.8
11.8 1.8
32.6 1.4
31.8 1.1
10.9 0.8
10.8 1.0
7.5 0.6
8.2 0.7
22.9 0.3
3.0 0.3
14.2 0.5
17.2 0.6
2.8 0.8
3.7 1.0
286 29
243 26
20.1 0.8A
17.2 1.9
2899.0 127.4
2702.7 163.6
10212.9 671.6
9482.6 850.0
44.5 2.6
43.3 2.0
15.2 3.3
16.9 2.0
8.1 2.0
6.6 1.6
36.2 2.3
33.9 1.6
13.2 1.1
13.1 0.9
9.1 0.7
9.7 0.8
4.1 0.5
3.6 0.2
15.6 0.8
17.6 0.9
3.0 0.9
4.1 1.2
346 42
250 29
20.6 2.3A
17.4 2.4
3322.8 206.6
3370.1 235.8
10985.5 894.7
10051.3 1058.5
48.1 2.8
47.0 2.4
17.0 1.7
19.8 1.6
9.1 1.3
8.9 1.5
32.5 2.4
31.5 1.8
10.6 1.2
11.1 0.9
8.2 1.0
8.4 0.6
4.7 0.9
4.2 0.5
15.4 0.6
16.8 0.7
3.0 1.2
4.1 1.5
269 29
469 38
27.6 3.1B
20.8 2.0
3524.4 288.0
3366.0 231.7
9556.1 495.2
9557.0 679.1
44.1 1.6
46.5 1.9
15.7 1.1
18.7 1.5
7.8 1.1
8.1 1.3
33.6 1.1
31.0 1.1
13.0 0.8
11.3 0.5
10.0 0.8
8.5 0.5
3.6 0.2
3.4 0.2
15.1 0.5
16.6 0.3
6.6 1.6
4.9 1.3
273 21
243 30
20.2 1.5A
16.9 1.6
3325.4 266.6
3171.0 283.6
9900.9 557.6
10470.5 1247.4
45.7 2.8
45.8 3.8
17.2 2.6
15.1 2.0
7.1 1.4
7.7 1.3
31.9 1.9
2.8 2.9
11.3 1.4
11.8 1.3
8.3 1.2
9.2 1.7
3.2 0.5
3.9 0.5
15.4 0.4
15.8 0.9
6.5 1.9
4.8 2.3
293 51
325 57
22.9 1.7
22.0 2.5
2803.2 245.1
2632.5 251.0
0.64
0.95
0.27
0.71
0.23
0.21
0.35
0.19
0.52
0.70
0.22
0.35
0.21
0.67
0.10
0.09
0.51
0.54
0.07
0.96
0.56
0.54
0.05
0.34
0.20
0.11
Ð
0.44
Ð
0.05c
Ð
0.12
Ð
0.69
Ð
0.38
Ð
0.36
Ð
0.16
Ð
0.58
Ð
0.20
Ð
0.02d
Ð
0.11
Ð
0.19
Ð
0.77
Data are means s.e.m. n ˆ 61. StLa, Streptococcus thermophilus ‡ Lactobacillus acidophilus; PY, placebo yoghurt; StLr, Streptococcus
thermophilus ‡ Lactobacillus rhamnosus; G, GAIO1; PP, placebo pill.
a
ANOVA for group difference in week 0 and week 8, respectively. bANOVA for group differences of changes from week 8 7 week 0. cStLa and PY 6ˆ G.
d
StLa, PY and StLr 6ˆ G.
Different letter (A,B): groups differ, P < 0.05.
European Journal of Clinical Nutrition
Probiotic milk products and cardiovascular risk
L Agerholm-Larsen et al
the strains Streptococcus thermophilus (St) and Enterococcus faecium (Ef). In vitro studies have shown that St is acidsensitive and cannot survive the passage through the small
intestine (Havenaar & Minekus, 1996). However, Ef is
know to have a good bile tolerance (Taranto et al, 1996).
Consequently, we suggest that Ef is the bacteria strain with
the cholesterol-lowering effect. This is also based on the
lack of effect on cholesterol of the two other bacteriacontaining yoghurts (StLa and StLr). They did not contain
Ef. The speci®c action of the strain is still not clear, but
probably relates to its in vitro ability to tolerate bile and
assimilate cholesterol (MD Foods Report, 1998, p 12).
The present hypocholesterolaemic effect of GAIO1
supports the results from previous studies on GAIO1. In
one study, consumption of 200 ml GAIO1 daily for 6
weeks reduced LDL-cholesterol in normal-weight men by
10% (Agerbñk et al, 1995). Our ®ndings are also supported
by the results of the recently publicised study by Bertolami
et al (1999). They found that LDL-cholesterol decreased by
6% after 8 weeks intake of 200 ml yoghurt in 32 subjects.
In another longer-term study (6 months) in normal-weight
men and women, a reduction in LDL-cholesterol by 8%
was observed after 1 month, but after 6 months, similar
reduction was observed in both the GAIO1 and the placebo
group (Richelsen et al, 1996). The latter study, however,
failed to ®nd a prolonged effect on cholesterol levels. A
potential reason is a reduction in compliance over time,
which should be monitored in future longer-term studies.
We expected to see differences in lipids after 4 weeks as
demonstrated in previous comparable studies. However, we
observed no tendencies towards a reduction of LDL-cholesterol in week 4. We have no obvious explanation for this
®nding. A possible mechanism behind this ®nding could
perhaps be the small numbers of subjects in each group. It
is likely that there is some between-subject variability in
intestinal colonisation of the active bacteria.
The reason for the ®nding of a negative correlation
between changes in LDL-cholesterol and changes in body
weight is not clear. It is likely that a slight change in dietary
composition induced by the yoghurt consumption is
responsible for this change, because we did not ®nd this
in the PP group, in which a slight increase occurred both in
body weight and in LDL-cholesterol. However, in accordance with the expected change we found a negative
correlation between change in HDL-cholesterol and
change in body weight.
Our study had an inbuilt enhanced compliance as the
participants had to consume 300 ml test-yoghurt three times
per week at the department under supervized ingestion. In
Dietary records
Complete dietary records were supplied by 61 subjects. The
average energy and macronutrient intake from the 7-day
weighed habitual dietary record in week 0 or 8 did not
differ between the groups (Table 5). When analysing the
changes from baseline (week 8 ± week 0), the change in
intake of alcohol was different between groups (group
effect: P ˆ 0.02; Table 5).
Compliance
Of all the breath tests the subjects did not carry out 3.6%
either because (a) they forgot to do it in time, (b) they did
not come to the department at the right time for collection
of the breath bags and enriched yoghurt, or (c) they
returned the bags too late for analysis. Signi®cant differences in the average DOB-values were found between the
PP-group compared to the yoghurt consuming groups
(P < 0.05; Table 6). This was expected since the DOBvalues for the PP-group should be 0% because no 13Cacetate was given. The results in the yoghurt-consuming
groups varied between 78.6% and 100% compliance
according to DOB-values above 6% (Table 6). No differences between the yoghurt-consuming groups were found.
However, signi®cant differences were found between men
and women in mean DOB-values (P < 0.05; data not
shown). Thus, during weeks 4, 6 and 8 men had a
signi®cantly lower mean DOB-value than women. This
was seen both when including and excluding the results
of the PP-group.
Discussion
The major ®nding of the present study was a decrease from
baseline in LDL-cholesterol after 8 weeks of GAIO1
consumption. The decrease in LDL-cholesterol amounted
to 0.26 0.1 mmol=l (a reduction of 8.4%) after adjustment
for changes in body weight. However, the differences in
changes in LDL-cholesterol between the G-group and the
other groups were only borderline signi®cant (P ˆ 0.09),
unless we compared only with the placebo groups
(P ˆ 0.02). When we compared all ®ve treatment groups,
unadjusted for changes in body weight, no statistical effects
were observed in week 8 in the G-group on LDL-cholesterol (P ˆ 0.29).
The reason for the observed hypocholesterolaemic effect
is proposed to be related to the bacteria culture in the
product (Agerbñk et al, 1995). A potential explanation
could be an association between gut micro¯ora and cholesterol absorption in the small intestine. GAIO1 contains
Table 6
Compliance (delta over baseline)
Week
2
4
6
8
295
StLa
DOB
% > 6%
DOB
% > 6%
DOB
% > 6%
DOB
% > 6%
PY
A
20.4 2.4
93.7
16.6 3.2A
81.2
18.1 2.0A
93.3
20.2 2.6A
85.7
StLr
A
18.7 2.2
92.9
17.1 1.7A
100
19.6 3.2A
92.9
18.5 2.3A
92.3
G
A
19.5 2.4
92.9
14.4 2.4A
78.6
16.2 1.5A
100
16.8 1.8A
100
PP
A
16.7 2.4
87.5
17.2 2.6A
81.2
17.7 2.1A
100
15.2 2.6A
85.7
B
7 0.6 0.2
0
7 0.5 0.1B
0
7 0.4 0.1B
0
7 0.4 0.2B
0
n
ANOVA P-value
70
P < 0.001
67
P < 0.001
67
P < 0.001
64
P < 0.001
Data are means s.e.m. StLa, Streptococcus thermophilus ‡ Lactobacillus acidophilus; PY, placebo yoghurt; StLr, Streptococcus
thermophilus ‡ Lactobacillus rhamnosus; G, GAIO1; PP, placebo pill. n ˆ 70. Missing values were excluded from analyses. DOB: delta over baseline.
6%, promille level for DOB values.
Different letter (A,B): groups differ, P < 0.05.
European Journal of Clinical Nutrition
Probiotic milk products and cardiovascular risk
L Agerholm-Larsen et al
296
addition, they did a compliance-test at home every second
week by consuming a 13C-acetate enriched yoghurt or a
placebo pill followed by a breath test. We conclude from
the latter that the study showed an acceptable compliance
since this varied from 78.6% to 100% and there were no
differences among the four yoghurt-consuming groups.
Another way of measuring dietary compliance is by a
lithium-marker technique. Lithium should be added to all
test products during manufacturing and then recovery
should be measured in the urine (Sanchez-Castillo et al,
1987; Alles et al, 1996; Heijnen et al, 1998). This compliance test could be used to advantage in future long-term
studies with milk products, since all products are marked
and not only one product of certain periods. The recovery
of lithium can anytime show if non-compliance could have
an important in¯uence on the outcome of the study's test
results. Possibly, this type of test could be an even better
detector of compliance than the one used here, provided
there is complete urine collection.
According to the dietary records, no differences between
the groups and no changes from baseline in the habitual
diet were found. Normal individual variation and the multiple testing can probably explain the small group differences
in changes of alcohol and carbohydrate intakes.
As placebo products we decided to use both a placebo
yoghurt based on a chemically fermented low-fat milk and
a placebo pill based on calcium lactate with a very low
calcium content. We included both placebo types to avoid
the possibility that a placebo, low-fat milk product without
the bacteria culture by itself could have cholesterol-lowering effects. We did not observe any effect of the placebo
products on LDL-cholesterol.
GAIO1 was associated with lower LDL-cholesterol and
higher ®brinogen concentrations than the placebo products.
In analyses involving only the three fermented yoghurts, we
were not able to show a group difference in LDL-cholesterol. Therefore, we can neither exclude nor support the
possibility that the alternative yoghurts StLa and StLr could
have bene®cial effects on LDL-cholesterol.
A high plasma ®brinogen concentration is a risk factor
of CVD in healthy men (Wilhelmsen et al, 1994; Heinrich
et al, 1994). Fibrinogen is an acute-phase protein and we
therefore speculate whether the increased ®brinogen concentration found in the G-group may be attributed to
immunostimulation. This has previously been described
mainly with strains belonging to the Lactobacillus gene,
although strains of Streptococcus thermophilus have been
examined too (Gabay & Kushner, 1999 (review)). However, one should note that the ®brinogen levels remained
within normal ranges (4.5 ± 10.3 mmol=l). C-reactive protein is another and more frequently used indicator of acutephase responses. In order to assess whether the observed
®brinogen-raising effect of the G-group could be part of an
acute-phase reaction, we therefore also assessed CRP in our
samples. CRP values were mostly beneath the detection
limit of the method, and the CRP determinations therefore
did not bring us more close to explain what caused the
signi®cant increase in ®brinogen in the G-group. We cannot
exclude from our present observations that a transient
colonic in¯ammation caused by bacteria strains in the
GAIO1 is the reason for the increase in ®brinogen in
subjects consuming GAIO1. The lack of any increase in
CRP does not, however, support this possibility.
We instructed the subjects to be weight-stable and not to
gain or lose more than a maximum of 2 kg body weight
European Journal of Clinical Nutrition
during the intervention. Despite this, all groups Ð even the
placebo pill group Ð gained some weight during the 8
weeks. One reason could be that the subjects were looking
forward to the slimming course at the end of the intervention and therefore were more relaxed about their weight.
This could not, however, be explained by changes in food
intakes since the energy intake and macronutrient intake Ð
according to the dietary records Ð was the same in weeks 0
and 8. General inaccuracies in dietary records could explain
this.
Conclusion
The CAUSIDO1 culture was associated with decreased
LDL-cholesterol and increased ®brinogen in overweight
subjects after daily consumption for 8 weeks. The decrease
in LDL-cholesterol amounted to 8.4%, being very similar
to the two previous Danish studies (about 10% and 8%).
Such a decrease in LDL-cholesterol would correspond to a
decrease in the risk of CVD of 20 ± 30% (Law & Wald,
1994) and is therefore clinically very relevant. The
increased level of ®brinogen could be a transient acutephase response caused by immunostimulation by Enterococcus faecium or Streptococcus thermophilus of the
GAIO1 product. The present results do not support that
in vitro properties can predict in vivo effects with respect to
LDL-cholesterol lowering effects of different bacteria
strains. These have to be validated in humans.
Acknowledgements ÐThe authors gratefully thank our colleague Peter
Marckmann for valuable discussions during the preparation of the manuscript and dietician Jette Rau, laboratory technician Kirsten Bryde Rasmussen, Berit Hoielt and her kitchen staff for expert technical assistance.
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