EXTENDED ABSTRACT

Double benefit claims for antimicrobial and antioxidative probiotic

MARIKA MIKELSAAR1, PIRJE HUTT1, TIIU KULLISAAR2, KERSTI ZILMER2

& MIHKEL ZILMER2

1Department of Microbiology, 2Department of Biochemistry, University of Tartu, Tartu, Estonia

AbstractThis paper describes the in vitro assessment of the functional properties of probiotic Lactobacillus fermentum ME-3(DSM14241) and evaluates the possibility for enhancement of the antioxidative activity of the human organism colonized ornot with Helicobacter pylori (Hp) through the use of the synbiotic comprising L. fermentum ME-3, Lactobacillus paracasei8700:2 and Bifidobacterium longum 46 with Raftilose P95 in a randomized double-blind placebo-controlled volunteer cross-over trial. In vitro the probiotic strain ME-3 suppressed both clinical and reference strains of Hp and expressed the highestantioxidative activity among the probiotic bacteria. In a volunteer trial the consumption of the synbiotic suppressed theoxidative stress indices in healthy volunteers colonized or not with Hp. However, this beneficial effect was apparent withoutsystemic antimicrobial influence of probiotic bacteria on Hp colonization. This suggests the possibility to apply the probioticstrain ME-3 with double health claims for reduction of the increased risk of cardiovascular diseases (CVD) in thecomposition of the synbiotic as an adjunct to Hp antimicrobial therapy.

Key words: Helicobacter pylori, antioxidative activity, probiotic, lactobacilli, bifidobacteria, oxidative stress, synbiotic

Introduction

Several gut microbes are elaborated for use as

probiotics in functional food to prevent and treat

various infections and other diseases. Probiotics

normalize the composition of the intestinal micro-

biota and modulate the immune and metabolic

response of the host (1). There are a few data related

to the systemic impact of probiotics in defence

against oxidative stress (OxS) -related diseases.

Internationally accepted functional food, offering

support against infections and also cardiovascular

diseases, has won popularity throughout the world.

The evidence-based claims comprise either enhance-

ment of particular human physiological functions or

reduction of some disease risk (2). However, there is

a clear need for combined in vitro assays and clinical

trials (3).

Helicobacter pylori (Hp) infection is associated with

gastritis and peptic ulcer disease, yet recent studies

have related chronic Hp infection to the pathogen-

esis of cardiovascular diseases (CVD) due to the

systemic impact (4�6). Probiotics in combination

with antibiotic treatment have been reported to be

successful in the management of Hp infection (7).

The probiotic Lactobacillus fermentum strain ME-3

(DSM-14241) has been elaborated according to the

regulations of WHO/FAO (2002) evolving the iden-

tification and molecular typing of this probiotic

strain of human origin, its deposition in an interna-

tional culture collection, and safety assessment by

laboratory tests and testing on experimental animals

and volunteers. It has been established that L.

fermentum strain ME-3 has double functional prop-

erties: antimicrobial activity against intestinal patho-

gens and physiologically relevant total antioxidative

activity (TAA) and total antioxidative status (TAS)

of intact cells and lysates (8).

This paper describes the in vitro assessment of the

functional properties of strain ME-3 against Hp

strains and evaluates the possibility for enhancement

of the antioxidative activity of subjects colonized or

not with Hp through the use of the synbiotic

comprising L. fermentum ME-3, Lactobacillus para-

casei 8700:2 and Bifidobacterium longum 46 with

Correspondence: Professor Marika Mikelsaar, Department of Microbiology and Department of Biochemistry, University of Tartu, Ravila str.19, Tartu 50411,

Estonia. Tel: �372 7 374179. Fax:�372 7 374172. E-mail: marika.mikelsaar@ut.ee

Microbial Ecology in Health and Disease. 2008; 20: 184�188

ISSN 0891-060X print/ISSN 1651-2235 online # 2008 Informa UK Ltd.

DOI: 10.1080/08910600802408178

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Raftilose P95 in a cross-over randomized double-

blind placebo-controlled volunteer trial.

Materials and methods

In vitro assays

The antimicrobial effect of L. fermentum ME-3 cells

against H. pylori NCTC 11637 and two clinical

isolates (HP8, HP9) was assessed (9). Antagonistic

activity was investigated on Columbia agar base

supplemented with 7% horse blood and 1% Vitox

agar plates using: (i) the streak line method and (ii)

inhibition zone assay at 378C in microaerobic con-

ditions. The growth inhibition of Hp strains was

measured in millimetres.

Trial on volunteers

We tested whether Hp colonization of the stomach

may be associated with some CVD-related OxS

indices that could be reduced by eradication of Hp

with a mixture containing the antioxidative probiotic

strain ME-3. The aim was to evaluate some blood

and lipoprotein markers for OxS in healthy subjects,

assessing their colonization with Hp.

Study population. Healthy adult volunteers (49

women and 12 men) without gastric symptoms were

included in the randomized, double-blind placebo-

controlled cross-over synbiotic efficacy study, con-

ducted in the period February to June 2005 in Tartu,

Southern Estonia. Fifty-three subjects completed the

trial. Eight subjects were withdrawn from the study

for various reasons (withdrawal rate 13%).

The subjects (61 persons) were checked for

colonization with Hp at recruitment and after

completing the cross-over study. The inclusion

criteria were: the wish to participate, age 20�60

years, no known health problems, and no consump-

tion of probiotic products or special diets. All the

participants signed the written informed consent and

were told that they could withdraw from the study at

any time. The exclusion criteria included a history of

gastrointestinal disease, food allergy and acute in-

fection; use of any antimicrobial agent within the last

month or use of any regular concomitant medication

including non-steroidal anti-inflammatory drugs and

antioxidant vitamins; and pregnancy or breastfeed-

ing. The Ethical Committee of Tartu University

approved the study protocol and the trial was

registered ISRCTN43435738.

Encapsulated bacteria

Multistrain capsules containing probiotic bacteria L.

paracasei 8700:2 (DSM 13434; Probi, Lund,

Sweden) and B. longum 46 (University of Turku,

Finland), obtained from the culture collection of the

University of Turku and probiotic L. fermentum ME-

3 (DSM 14241), obtained from the culture collec-

tion of Tartu University, were used.

All included strains of bifidobacteria and lactoba-

cilli inhibited the growth of the H. pylori reference

strain (NCTC 11637), and bifidobacteria expressed

the highest activity (9).

Study design

Volunteers were randomly allocated to receive en-

tero-coated capsules containing the freeze-dried

probiotic 3�109 cfu and one sachet of a well-known

prebiotic (6.6 g Raftilose P95; Orafti) or placebo

(maltodextrin) twice a day for 3 weeks. After a 2

week washout period, volunteers were crossed over

to another 3 weeks of synbiotic/placebo administra-

tion.

Samples of fasting blood and faecal samples were

collected at recruitment. All subjects were checked

for colonization by Hp. In faecal samples the

presence of the Hp antigen was determined by the

HpSA test (ImmunoCard STAT HpSA, Meridian

Bioscience Europe, Italy) (10,11).

Antioxidative activity of consumed strains and human

sera

The total antioxidative activity (TAA) and total

antioxidative status (TAS) of probiotic strains and

a 3% solution of Raftilose P95 as well as blood sera

of participants were measured.

Briefly, lactobacilli were grown in MRS broth

under microaerobic conditions and bifidobacteria

in MRS broth supplemented with cysteine in an

anaerobic environment for 48 h. Probiotic bacteria

were then pelleted by centrifugation (10 000 g for 10

min), washed twice and resuspended in isotonic

saline at 48C. The density of suspension was

adjusted to 109 cfu ml�1 using an absorbance of

1.1 at 600 nm.

TAA was assessed by the linolenic acid test (LA

test) as described previously (8,12). This test eval-

uates the ability of the sample to inhibit lipid

peroxidation. TAS was measured with a commer-

cially available kit (TAS, Randox Laboratories Ltd,

Ardmore, UK), with water-soluble vitamin E (Tro-

lox) serving as a standard. This method is based on

the inhibition of the absorbance of the ferrylmyoglo-

bin radicals of 2,2?-azinobis-ethylbenzothiazoline 6-

sulfonate (ABTS�) generated by activation of

metmyoglobin peroxidase with H2O2 (13). TAA

was expressed as the inhibition percentage of the

peroxidation of linolenic acid standard by the

sample. The total antioxidative values of probiotic

Antimicrobial and antioxidative probiotic 185

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bacteria were considered high if TAA was arbitrarily

�20% and TAS �0.1 mmol/l.

Baseline diene conjugates of LDL particles

Baseline diene conjugates of LDL particles (BDC-

LDL) were measured by the method described by

Ahotupa et al. (14) using spectrophotometric mea-

surements. Absorbance units (difference A234 �A300)

were converted to molar units using the molar

extinction coefficient 2.95�104 M�1 cm�1. For

BDC-LDL, the coefficient of variation (CV) for

within-assay precision was 4.4%, and the CV for the

between-assay precision over a period of 3 months

was 4.5%.

Oxidized LDL level

Oxidized LDL (oxLDL) level was measured in

serum (15) using the Mercodia Oxidized LDL

Enzyme-Linked Immunosorbent Assay (ELISA) kit

(Cat. No. 10-1143-01; Mercodia AB, Uppsala,

Sweden). Mercodia Oxidized LDL ELISA is a

solid-phase two-site enzyme immunoassay, based

on the direct sandwich technique, in which two

monoclonal antibodies are directed against separate

antigenic determinants on the modified apolipopro-

tein B molecule of LDL.

Statistics

The computer program Sigma Stat for Windows 2.0

(Jandel Corporation, USA) was applied. Baseline

and intervention data were compared by using

paired t test or Student’s t test or the Mann-Whitney

rank sum test. The selection of tests was made

automatically according to the distribution of the

data. All data were given as mean and standard

deviation (SD). Differences were considered statis-

tically significant if the p value wasB0.05.

Results and discussion

In vitro study

Antagonistic activity of L. fermentum ME-3 was

significantly higher (pB0.005) against clinical iso-

lates of Hp than the reference strain (Table I). L.

fermentum ME-3 expressed the highest values of

antioxidative activity according to both applied

methods in comparison with the other probiotic

strains. Only the TAS values of B. longum 46 did not

differ significantly from those of L. fermentum ME-3

(p�0.082). The 3% solution of Raftilose P95 did

not show an antioxidative activity according to the

methods used (Table II).

Volunteer study

Outcome. In all, 53 subjects (12 males and 41

females) out of 61 completed the trial. Eight subjects

(13%) were withdrawn from the study for various

reasons, including non-compliance (n�1), preg-

nancy (n�2), acute infection and antimicrobial

treatment (n�2), diarrhoea (n�1), rheumatic fever

(n�1) and heart arrhythmia (n�1).

Although the synbiotic was well tolerated by all

subjects, 9 of 53 (17%) reported mild and 2 (4%)

persons moderate abdominal pain. Six subjects

reported increased flatulence and 16 increased stool

frequency during administration of synbiotic.

Treatment results of Hp infection. There was a

quite similar distribution of Hp-positive (n�28,

53%) and Hp-negative (n�25, 47%) subjects,

which did not change after the treatment.

Antioxidative indices. After synbiotic consump-

tion, the mean values of TAA increased significantly

(41 vs 42%, pB0.001) and the values of BDC-LDL

and oxLDL decreased (15.2 vs 12.7 mM/l, pB0.001;

132.5 vs 122.8 U/l, p�0.047, respectively) as

compared with baseline values (Table III). Hp-

colonized volunteers had lower TAS values (B1.0

mmol/l) (OR�5.7 (1.79�20.24)).

After synbiotic consumption the values of BDC-

LDL decreased in both Hp-positive and Hp- nega-

tive subjects. The TAA and TAS values improved in

Hp-positive subjects, although the TAS still stayed

Table I. Antagonistic activity of L. fermentum ME-3 against H.

pylori strains.

H. pylori strains Inhibition zone (mm)

Median/range Mean9SD

H. pylori NCTC 11637 14/13�21 15.592.7

HP 8 17/12�25 17.893.3

HP 9 19/15�30 20.495.0

Hp NCTC 11637 vs Hp 9 (pB0.005).

Table II. Antioxidative activity of probiotic strains and prebiotic

(mean9SD) in vitro.

Strains TAA (%) TAS (mmol/l)

L. fermentum ME-3 2494* 0.1890.05$,%L. paracasei 8700:2 1594* 0.0390.03$B. longum 46 1194* 0.1090.08%Raftilose P95 0 0

TAA, total antioxidative activity; TAS, total antioxidative status.

*TAA values: L. fermentum strain ME-3 vs L. paracasei 8700:2, L.

fermentum ME-3 vs B. longum 46 (pB0.05).

$TAS values: L. fermentum ME-3 vs L. paracasei 8700:2 (pB

0.05).

%TAS values: L. fermentum ME-3 vs B. longum 46 (p�0.082).

186 M. Mikelsaar et al.

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lower in Hp-colonized persons than in these not

colonized (Table IV).

In this study we confirmed the hypothesis that the

consumption of the synbiotic, comprising L. fermen-

tum ME-3, L. paracasei 8700:2 and B. longum 46

with Raftilose P95 can improve OxS-related indices

in healthy volunteers when half of them were

colonized with Hp. However, this beneficial effect

was apparent without systemic antimicrobial influ-

ence of probiotic bacteria on Hp colonization.

Recent studies have shown the suppressive effect

of the antioxidative probiotic L. fermentum ME-3 on

CVD-related OxS indices in healthy subjects

(12,15). However, still, there has been a lack of

studies with different combinations of probiotic

strains and prebiotics on the OxS in human body.

Although all three selected probiotics had some

antioxidative activity, the main antioxidant com-

pound in the synbiotic mixture was still L. fermentum

ME-3 expressing potent TAA and TAS of intact cells

and lysates (8). The combined effect was also

possible as the fructo-oligosaccharide-based prebio-

tic raftilose could modulate the gut microbiota of

humans, increasing the production of short chain

fatty acids (SCFAs) capable of reduction of the

production of reactive oxygen species in the gut (16).

Previously, probiotic strain ME-3 has expressed

moderate antimicrobial activity against the Hp

reference strain (9) and in the current study it even

more effectively suppressed the clinical isolates of

Hp. Moreover, some antimicrobial peptides from

strain ME-3 lysates have been assessed by MALTI-

TOF MS (17). Seemingly, in the entero-coated

capsule the direct impact of lactobacilli cells on Hp

was not possible and the putative antimicrobial

compounds did not reach the gastric or duodenal

mucosa colonized by Hp via the lymphatic or hepatic

blood circulation.

In contrast, the systemic enhancement of the

human organism’s antioxidative activity (TAA and

TAS) by synbiotic containing strain ME-3 was well

expressed in blood. Nevertheless, the values of TAS

indicating the antioxidative activity in water-related

fractions, dominated by plasma albumin, stayed

lower in Hp-colonized subjects. This corresponds

to the data for reduced serum albumin values in Hp-

colonized persons (18). Hp infection has been

shown to exert both local and systemic OxS in

patients suffering from peptic ulcer (19). In healthy

volunteers colonized by Hp the antioxidative status

of blood has been significantly lower than in those

not colonized (20). The OxS, classified as imbalance

between oxidants and antioxidants, in recent years

has mainly been recognized as a process with

implications for many pathophysiological events

(21). The systemic OxS and reduced antioxidative

capacity of blood sera might collaborate in the

increased risk for developing CVD. Also, after

synbiotic consumption the bioquality of LDL parti-

cles assessed by baseline diene conjugates of LDL

and important in the development of CVD was

improved in all volunteers. However, these beneficial

Table III. Oxidative stress-related indices in blood after consumption of the synbiotic (paired t test).

Parameter Baseline After synbiotic After placebo Baseline vs synbiotic Baseline vs placebo Synbiotic vs placebo

TAA 4192 4292s 4292 B0.001 0.019 NS

TAS 1.0190.10 1.0490.11 1.0590.09 0.004 B0.001 NS

BDC-LDL 15.296.1 12.794.1 14.697.3 B0.001 0.068 0.035

oxLDL 132.5950.5 122.8945.6 118.3947.6 0.047 0.009 NS

TAA, total antioxidative activity; TAS, total antioxidative status; BDC-LDL, baseline diene conjugates of LDL particles; oxLDL, oxidized

LDL; NS, not significant.

Table IV. H. pylori colonization and oxidative stress-related indices of blood.

Parameter Hp-negative Hp-positive

Baseline After synbiotic Baseline After synbiotic p values

BDC-LDL 14.895.4 12.693.8 15.696.8 12.794.4 B0.001* 0.004$oxLDL 134.0957.6 121.6949.7 131.1944.3 123.9942.5 NS

TAA 4292 4292 4192 4292 B0.001$TAS 1.0590.10 1.0690.13 0.9790.10 1.0390.10 0.014% B0.001$

TAA, total antioxidative activity; TAS, total antioxidative status; BDC-LDL, baseline diene conjugates of LDL particles; oxLDL, oxidized

LDL; NS, not significant.

*Hp-negative baseline vs synbiotic.

$Hp-positive baseline vs synbiotic.

%Hp-positive vs Hp-negative.

Antimicrobial and antioxidative probiotic 187

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effects were not accompanied by the systemic anti-

microbial influence of probiotic bacteria on Hp

colonization.

The consumption of the synbiotic, comprising a

prebiotic with lactic acid bacteria, particularly the

antimicrobial and antioxidative probiotic L. fermen-

tum ME-3, can suppress oxidative stress indices in

healthy volunteers. This suggests the possibility to

apply the probiotic strain ME-3 with double health

claims for reduction of the increased risk of CVD in

the composition of the synbiotic as an adjunct to Hp

antimicrobial therapy.

Acknowledgements

The study was supported by a grant from the

Estonian Science Foundation (basic funding

0182555s03 and 6588) and EU Research Commis-

sion Grant QLRT-2001-00135. We thank our EU

partners for excellent cooperative research.

Competing interests: Lactobacillus fermentum

ME-3 and/or its use as a probiotic has been patented

(Estonian patent EE4580, US patent US7244424,

Russian Federation patent RU2284354, European

patent application EP1401457, international patent

application WO03002131). The owner of the pa-

tents is the University of Tartu. The authors are

being rewarded proportionally according to their

contribution towards the creation of intellectual

property in accordance with the Order of Handling

Inventions valid at the University of Tartu.

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