Journal of Applied Microbiology 2003, 94, 981–987

Adhesion and aggregation ability of probiotic strain

Lactobacillus acidophilus M92

B. Kos1, J. Šušković1, S. Vuković2, M. Šimpraga2, J. Frece1 and S. Matošić1

1
Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia, and 2Veterinary Faculty, University of Zagreb, Croatia

2002/257: Received 2 July 2002, revised 23 January 2003 and accepted 29 January 2003

ABSTRACT
B . K O S , J . Š U Š K O V I Ć , S . V U K O V I Ć , M . Š I M P R A G A , J . F R E C E A N D S . M A T O Š I Ć . 2003.
Aims: To investigate aggregation and adhesiveness of Lactobacillus acidophilus M92 to porcine ileal epithelial cells
in vitro, and the influence of cell surface proteins on autoaggregation and adhesiveness of this strain.
Methods and Results: Lactobacillus acidophilus M92 exhibits a strong autoaggregating phenotype and manifests a
high degree of hydrophobicity determined by microbial adhesion to xylene. Aggregation and hydrophobicity were
abolished upon exposure of the cells to pronase and pepsin. Sodium dodecyl sulphate polyacrylamide gel
electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins,
approximated at 45 kDa, in L. acidophilus M92. The relationship between autoaggregation and adhesiveness to
intestinal tissue was investigated by observing the adhesiveness of L. acidophilus M92 to porcine ileal epithelial cells.
Removal of the S-layer proteins by extraction with 5 mol l)1 LiCl reduced autoaggregation and in vitro adhesion of
this strain.
Conclusions: These results demonstrate that there is relationship between autoaggregation and adhesiveness
ability of L. acidophilus M92, mediated by proteinaceous components on the cell surface.
Significance and Impact of the Study: This investigation has shown that L. acidophilus M92 has the ability to
establish in the human gastrointestinal tract, which is an important determinant in the choice of probiotic strains.

Keywords: adhesion, aggregation, hydrophobicity, Lactobacillus acidophilus, probiotic properties, surface layer
proteins.

enable specific interactions between adhesins (usually pro-

INTRODUCTION
teins) and complementary receptors (Freter 1992; Rojas and
Adhesion to intestinal epithelial cells is an important pre- Conway 1996; Pérez et al. 1998). Autoaggregation of
requisite for colonization of probiotic strains in the gastro- probiotic strains appeared to be necessary for adhesion to
intestinal tract, preventing their immediate elimination by intestinal epithelial cells, and coaggregation abilities may
peristalsis and providing a competitive advantage in this form a barrier that prevents colonization by pathogenic
ecosystem (Pedersen and Tannock 1989; Freter 1992; Alander microorganisms (Reid et al. 1988; Boris et al. 1997; Del Re
et al. 1997). Difficulties involved in studying bacterial et al. 2000). Physicochemical characteristics of the cell
adhesion in vivo, especially in humans, have led to the surface such as hydrophobicity may affect autoaggregation
development of in vitro model systems for the preliminary and adhesion of bacteria to different surfaces (Wadström
selection of potentially adherent strains (Mäyrä-Mäkinen et al. 1987; Pérez et al. 1998; Del Re et al. 2000). The
et al. 1983, Conway and Kjellberg 1989; Kimoto et al. 1999). proteinaceous nature of some surface components has been
Bacterial adhesion is initially based on non-specific demonstrated, and surface layer (S-layer) proteins detected
physical interactions between two surfaces, which then in some Lactobacillus strains may be involved in adherence
(Schneitz et al. 1993; Mukai and Arihara 1994).
Correspondence to: Blazˇenka Kos, Faculty of Food Technology and Biotechnology, Lactobacillus acidophilus is one of the major species of this
University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia (e-mail: bkos@pbf.hr). genus found in human and animal intestines. When present
ª 2003 The Society for Applied Microbiology
982 B . K O S ET AL.

in sufficient numbers, as probiotics, the lactobacilli are assay. Equal volumes (2 ml) of each cell suspension were
believed to be able to create a healthy equilibrium between mixed together in pairs by vortexing for 10 s. Control tubes
beneficial and potentially harmful microflora in the gut were set up at the same time, containing 4 ml of each
(Tannock 1999; Šušković et al. 2001). The potential bacterial suspension on its own. The absorbance (A) at
probiotic strain L. acidophilus M92 has shown the ability 600 nm of the suspensions were measured after mixing and
to resist digestion processes in the gastrointestinal tract and after 5 h of incubation at room temperature. Samples were
is bile resistant (Kos et al. 2000; Šušković et al. 2000). taken in the same way as in the autoaggregation assay. The
Furthermore, in vitro studies have shown that L. acidophilus percentage of coaggregation was calculated using the
M92 can assimilate cholesterol in the presence of bile, so it is equation of Handley et al. (1987):
postulated that this strain might help in lowering serum ðð Ax þ AyÞ=2Þ  Aðx þ yÞ
cholesterol in vivo (Kos 2001). Coaggregation ð%Þ ¼  100
Ax þ Ay=2
The aim of this study was to investigate the aggregation
abilities and adhesive properties of L. acidophilus M92 to where x and y represent each of the two strains in the
porcine ileal epithelial cells in vitro, which were used because control tubes, and (x + y) the mixture.
of the similarity of porcine and human intestinal tracts. The
role of cell surface proteins on autoaggregation and adhe-
siveness of L. acidophilus M92 was also determined. Microbial adhesion to solvents
Microbial adhesion to solvents (MATS) was measured
according to the method of Rosenberg et al. (1980) with
M A T E R I A LS A N D M E T H O D S
some modifications (Crow and Gopal, 1995; Bellon-
Bacterial strains and growth conditions Fontaine et al. 1996). Bacteria were harvested in the
stationary phase by centrifugation at 5000 g for 15 min,
Lactobacillus acidophilus M92, Lactobacillus plantarum L4,
washed twice, and resuspended in 0Æ1 mol l)1 KNO3
Enterococcus faecium L3, Escherichia coli 3014 and Salmonella
(pH 6Æ2) to approximately 108 CFU ml)1. The absorbance
serotype Typhimurium from the culture collection of the
of the cell suspension was measured at 600 nm (A0). One
Department of Biochemical Engineering, University of
millilitre of solvent was added to 3 ml of cell suspension.
Zagreb and the standard strain L. acidophilus ATCC 4356
After a 10-min preincubation at room temperature, the two-
were used in this study. All lactic acid bacteria were stored at
phase system was mixed by vortexing for 2-min. The
)70C in de Man Rogosa Sharpe (MRS) broth (Difco,
aqueous phase was removed after 20 min of incubation at
Detroit, MI, USA) with 30% glycerol. Escherichia coli 3014
room temperature, and its absorbance at 600 nm (A1) was
and S. Typhimurium were maintained on nutrient agar
measured. The percentage of bacterial adhesion to solvent
(Difco) slopes at 4C. Before experimental use, cultures
was calculated as (1)A1/A0) · 100.
were subcultured twice in MRS or in nutrient broth (Difco).
Three different solvents were tested in this study: xylene
(Kemika, Zagreb, Croatia), which is an apolar solvent;
Autoaggregation and coaggregation assays chloroform (Kemika), a monopolar and acidic solvent; and
ethyl acetate (Kemika), a monopolar and basic solvent. Only
Autoaggregation assays were performed according to Del Re
bacterial adhesion to xylene reflects cell surface hydrophob-
et al. (2000) with certain modifications. Bacteria were grown
icity or hydrophilicity. The values of MATS obtained with
for 18 h at 37C with MRS solid or liquid medium. The
the two other solvents, chloroform and ethyl acetate, were
cells were harvested by centrifugation at 5000 g for 15 min,
regarded as a measure of electron donor (basic) and electron
washed twice and resuspended in their culture supernatant
acceptor (acidic) characteristics of bacteria, respectively
fluid or in phosphate buffered saline (PBS) to give viable
(Bellon-Fontaine et al. 1996).
counts of approximately 108 CFU ml)1. Cell suspensions
(4 ml) were mixed by vortexing for 10 s and autoaggregation
was determined during 5 h of incubation at room tempera- In vitro test for adhesion of L. acidophilus M92
ture. Every hour 0Æ1 ml of the upper suspension was to pig intestinal epithelium
transferred to another tube with 3Æ9 ml of PBS and the
The adhesion test was peformed using the method of Mäyrä-
absorbance (A) was measured at 600 nm. The autoaggrega-
Mäkinen et al. (1983), with modifications. Ileal samples were
tion percentage is expressed as: 1)(At/A0) · 100, where At
collected from 6-month-old Landras pigs. The tissues were
represents the absorbance at time t ¼ 1, 2, 3, 4 or 5 h and
held in PBS at 4C for 30 min to loosen surface mucus, and
A0 the absorbance at t ¼ 0.
then washed three times with PBS. The adhesion test was
The method for preparing the cell suspensions for
performed by incubating tissue samples (1 cm2) in bacterial
coaggregation was the same as that for autoaggregation
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 981–987
 ADHESION OF L. ACIDOPHILUS M92 983

suspensions (109 cells ml)1 PBS) at 37C for 30 min. Samples a strong autoaggregating phenotype. Better growth of the
of ileum were fixed in 10% formalin, dehydrated by increasing bacterium on MRS broth than on MRS agar could be the
concentrations of ethanol, and embedded in paraffin. Serial reason for slightly better autoaggregation of cells grown on
sections (5 lm) were cut, mounted on standard microscope MRS broth. The observed autoaggregation could be related
slides and stained for identification of Gram-positive and to cell surface component, because it was not lost after
Gram-negative bacteria, according to Brown and Brenn (Švob washing and suspending of the cells in PBS (Fig. 1).
1974). Slides were examined and photographed using a Nikon Coaggregation of L. acidophilus M92 with two other
Mikrophot-FXA light microscope (Nikon, Tokyo, Japan). potential probiotic strains (L. plantarum L4, Ent. faecium
L3) and two enteropathogens (S. Typhimurium, E. coli) was
also examined (Table 1). Results are expressed as the
Enzymic and chemical treatments
percentage reduction after 5 h in the absorbance of a mixed
of bacterial cells
suspension compared with the individual suspension.
Cultures were harvested by centrifugation (5000 g, 15 min), Lactobacillus acidophilus M92 demonstrated marked coag-
washed twice with distilled water and resuspended in the gregation with Ent. faecium L3 (Fig. 2), E. coli and
appropriate buffer. Cell suspensions, prepared as described S. Typhimurium (Table 1).
above, were subjected to different pH values of pronase,
pepsin, metaperiodate and LiCl. All chemicals were obtained
from Sigma (St Louis, MO, USA). The effect of different pH
80
values on hydrophobicity and autoaggregation ability of L.
acidophilus M92 was assayed in 0Æ1 mol l)1 citrate/phosphate 70
buffer, pH 2Æ8 and 0Æ1 mol l)1 acetate buffer, pH 4Æ5.
Pronase treatment (1 g l)1, 30 min at 37C) was carried out 60
in PBS, pH 7Æ2, pepsin treatment (0Æ5 g l)1, 30 min) in
Autoaggregation (%)

0Æ1 mol l)1 citrate/phosphate buffer, pH 2Æ8 and metaper- 50

iodate treatment (10 g l)1, 30 min) in 0Æ1 mol l)1 acetate
buffer, pH 4Æ5. The S-layer proteins of L. acidophilus M92 40
and L. acidophilus ATCC 4356 were removed by extraction
with 5 mol l)1 LiCl (30 min at 37C). Subsequently, 30
bacterial cell suspensions were washed twice and resuspended
20
in PBS for MATS, aggregation and adhesion assays.
10
SDS-PAGE of surface proteins from bacterial cells
0
Untreated bacteria or cells which had been extracted with 0 1 2 3 4 5
5 mol l)1 LiCl, as described above, were resuspended in 1% Time (h)
SDS for 30 min at 37C, for the isolation of surface proteins.
After centrifugation (9000 g, 5 min), the supernatant was Fig. 1 Comparison of the autoaggregation ability of Lactobacillus
acidophilus M92 cells resuspended in PBS (pH 7Æ2) after grown on
analysed by Sodium dodecyl sulphate polyacrylamide gel
MRS agar (j) and in MRS broth (m) or resuspended in their own
electrophoresis (SDS-PAGE) according to the method of
culture supernatant fluid (s). Error bars represent standard deviations
Laemmli (1970), using 10% polyacrylamide gels. Protein of the mean values of results from three replicate experiments
bands were visualized by staining the gels with Coomassie
brilliant blue. Low molecular weight markers from 14Æ4 to
97Æ0 kDa (a-lactalbumin, trypsin inhibitor, carbonic anhyd- Table 1 Coaggregation ability of Lactobacillus acidophilus M92 after
rase, ovalbumin, albumin and phosphorylase b) were obtained 5 h incubation at room temperature in PBS (pH 7Æ2)
from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Coaggregation with
Bacteria L. acidophilus M92 (%)

RESULTS Lactobacillus plantarum L4 4Æ36 (±1Æ81)

Enterococcus faecium L3 19Æ46 (±2Æ63)
Autoaggregation, coaggregation and Escherichia coli 3014 15Æ11 (±2Æ07)
adhesiveness of L. acidophilus M92 Salmonella serotype Typhimurium 15Æ70 (±2Æ98)

The sedimentation rate of L. acidophilus M92 was measured Mean (±standard deviation) of results from three separate experi-
over a period of 5 h. Results showed that the strain exhibited ments.

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 981–987
984 B . K O S ET AL.

Fig. 2 Coaggregation between Lactobacillus acidophilus M92 and

Enterococcus faecium L3 incubated in PBS (pH 7Æ2). Magnification,
·1000. Bar represents 10 lm

The adhesiveness of L. acidophilus M92 to the intestinal

tissue was also investigated. Microscopic examinations
showed that this species strongly adhered to porcine ileal
epithelial cells (Fig. 3).

Influence of cell surface properties

on autoaggregation and adhesion
of L. acidophilus M92
The MATS method was used to evaluate the hydropho- Fig. 3 Adhesion test with Lactobacillus acidophilus M92: intestinal
bic/hydrophilic cell surface properties of L. acidophilus epithelium of the pig before (a) and after treatment with suspension
M92 and compare them with the cell surface properties of of L. acidophilus M92 cells (b). Magnification, ·200. Bars represent
two other probiotic bacteria. The results indicated that 60 lm
L. acidophilus M92 was hydrophobic, whereas L. plantarum
L4 and Ent. faecium L3 were fully hydrophilic (Table 2). at pH 2Æ8, but pepsin treatment eliminated this property.
Bacterial adhesion to chloroform and ethyl acetate was The same effect on hydrophobicity was found with
tested to assess the Lewis acid–base characteristics of the pronase. Moreover, autoaggregation ability was moderately
bacterial cell surfaces. All strains showed stronger affinity reduced when L. acidophilus M92 was subjected to pronase
for chloroform, which is an acidic solvent and electron and pepsin activity. By oxidizing cell surface carbohydrates
acceptor, than for ethyl acetate, which is a basic solvent with metaperiodate, the cell surface hydrophobicity was
and electron donor (Table 2). Lactobacillus acidophilus M92 slightly affected, but autoaggregation of L. acidophilus M92
was further subjected to different treatments to character- was not significantly affected. When the surface proteins of
ize the cell surface components responsible for its hydro- L. acidophilus M92 were extracted and analysed by SDS-
phobicity and autoaggregation ability. Table 3 shows that PAGE (Fig. 4) the dominant protein band of L. acidophilus
the hydrophobicity of L. acidophilus M92 slightly decreased M92 had a molecular mass of approximately 45 kDa. After

Table 2 Adhesion of Lactobacillus acidophi-

Adhesion (%)
lus M92, L. plantarum L4 and Enterococcus
Bacteria Xylene Chloroform Ethyl acetate faecium L3 to xylene, chloroform and ethyl
acetate
Lactobacillus acidophilus M92 70Æ96 (±1Æ63) 36Æ06 (±1Æ28) 0
Lactobacillus plantarum L4 6Æ52 (±1Æ43) 47Æ03 (±1Æ84) 18Æ32 (±0Æ98)
Enterococcus faecium L3 0 61Æ21 (±2Æ02) 0

Mean (±standard deviation) of results from three separate experiments.

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 981–987
 ADHESION OF L. ACIDOPHILUS M92 985

Table 3 Effect of different treatments on cell surface hydrophobicity 75

and autoaggregation ability of Lactobacillus acidophilus M92

Cell surface 60

Autoaggregation (%)
Treatment hydrophobicity (%) Autoaggregation (%)

pH 7Æ2 70Æ96 (±2Æ11)a 71Æ30 (±2Æ08)a 45

pH 4Æ5 73Æ59 (±1Æ33)a 68Æ89 (±1Æ84)a
pH 2Æ8 55Æ85 (±1Æ47)b 62Æ14 (±1Æ27)b 30
Pronase 0 58Æ12 (±1Æ84)c
Pepsin 0 54Æ52 (±2Æ17)c
Metaperiodate 64Æ84 (±1Æ34)c 67Æ17 (±2Æ78)a 15

Values in parentheses represent the standard deviation. Mean values

0
(n ¼ 3) with no common superscript letters differ significantly 0 1 2 3 4 5
(P < 0Æ05). Time (h)

Fig. 5 Comparison of the autoaggregation ability of Lactobacillus

1 2 3 4 5 6 7 acidophilus M92 and L. acidophilus ATCC 4356 before (m M92, j
ATCC 4356) and after removal of their surface layer proteins with
5 mol l)1 LiCl (n M92, u ATCC 4356). Error bars represent standard
97 kDa deviations of the mean values of results from three replicate
66 kDa experiments
45 kDa

20·1 kDa
14·4 kDa

Fig. 4 SDS-PAGE of cell surface proteins from Lactobacillus acido-

philus ATCC 4356 and L. acidophilus M92 before and after extraction
with 5 mol l)1 LiCl; lanes 2, 3 and 4, L. acidophilus ATCC 4356 cell
surface proteins from untreated cells (lanes 2 and 4) and from cells
extracted with 5 mol l)1 LiCl (lane 3); lanes 5, 6 and 7, L. acidophilus
M92 cell surface proteins from untreated cells (lanes 5 and 7) and from
cells extracted with 5 mol l)1 LiCl (lane 6); lane 1, low molecular
weight protein standards

extraction of bacterial cells with 5 mol l)1 LiCl, more than

90% of S-layer proteins from both L. acidophilus M92 and
the control strain L. acidophilus ATCC 4356 were removed.
After S-layer removal, these two strains were tested for
autoaggregation. In both cases, removal of S-layer proteins
reduced their autoaggregation ability (Fig. 5). When the
contribution of these proteins in adhesiveness of
L. acidophilus M92 to pig intestinal epithelium was
examined, the results suggested that these proteins could
be involved in adhesion processes, because of weaker
adhesiveness of LiCl-treated bacterial cells in relation to
untreated cells (Fig. 6).

DISCUSSION
The ability to adhere to epithelial cells and mucosal surfaces Fig. 6 Adhesion of Lactobacillus acidophilus M92 to the intestinal
has been suggested to be an important property of many epithelium cells of the pig before (a) and after treatment of bacterial
bacterial strains used as probiotics. Cell adhesion is a cells with 5 mol l)1 LiCl (b). Magnification, ·600. Bars represent
multistep process involving contact of the bacterial cell 20 lm

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 981–987
986 B . K O S ET AL.

membrane and interacting surfaces. Several workers have surfaces are associated with the presence of polysaccharides
investigated the composition, structure and forces of (Green and Klaenhammer 1994; Rojas and Conway 1996;
interaction related to bacterial adhesion to intestinal epithe- Pelletier et al. 1997). It is known that only pronase- and
lial cells (Green and Klaenhammer 1994; Pelletier et al. pepsin-sensitive surface molecules are responsible for cell
1997; Pérez et al. 1998; Del Re et al. 2000). In most cases, surface hydrophobicity in bacteria. In this study, the
aggregation ability is related to cell adherence properties autoaggregation ability of L. acidophilus M92 was also
(Vandevoorde et al. 1992; Boris et al. 1997; Del Re et al. reduced by proteolytic treatment, while metaperiodate did
2000). Therefore, the potential probiotic strain L. acidophilus not much affect either hydrophobicity, or autoaggregation
M92 was examined for its autoaggregation ability and (Table 3). As bacterial cells subjected to proteolytic attack
coaggregation with some lactic acid bacteria and potential weaken their autoaggregation ability, it is reasonable to
pathogenic bacteria. Broth-grown and agar-grown cells of consider proteins as mediators in the aggregation process.
L. acidophilus M92, suspended in PBS (pH 7Æ2) were com- The SDS-PAGE of cell surface proteins of L. acidophilus
pared for their autoaggregation ability, because the method M92 revealed the presence of S-layer proteins, of an
of culture has been recognized as a factor that may affect approximate molecular mass of 45 kDa. The S-layer pro-
bacterial aggregation (Reid et al. 1992; Spencer and Chesson teins form a crystalline layer around many bacterial species,
1994). To avoid the possibility of removing extracellular amounting to 15–20% of the total cellular protein content,
components, which may have been concerned with autoag- apparent molecular weights of 40–200 kDa (Pouwels et al.
gregation, broth-grown cells were additionally examined for 1998; Sára and Sleytr 2000). It has been proposed that these
autoaggregation ability resuspended in their own culture proteins are involved in cell protection and surface recog-
supernatant fluid. Lactobacillus acidophilus M92 showed a nition, and that they could be potential mediators in the
strong autoaggregating phenotype which was not lost after initial steps involved in adhesion (Schneitz et al. 1993;
washing and suspending of the cells in PBS. To quantify Green and Klaenhammer 1994; Mukai and Arihara 1994).
interbacterial adherence, a coaggregation assay was devel- To assess the potential contribution of these proteins to
oped, which established coaggregation between L. acidophi- autoaggregation and adherence, bacterial cells were extracted
lus M92 and two other potential probiotic strains, with 5 mol l)1 LiCl to remove S-layer proteins. The results
particularly Ent. faecium L3, which could increase their showed that these proteins are important for autoaggrega-
colonization potential if they were to be used in mixed tion in L. acidophilus M92. Furthermore, adherence was
culture as probiotics. Furthermore, it has been suggested markedly different between non-treated bacterial cells and
that inhibitor producing lactic acid bacteria, which coaggre- cells treated with 5 mol l)1 LiCl (Fig. 6a, b). The reduction
gate with pathogens, may constitute an important host of adherence after removing S-layer proteins from the cell
defence mechanism against infection in the urogenital tract surface suggests that these proteins promoted the adhesive-
(Reid et al. 1988). Also, a similar protective mechanism ness of L. acidophilus M92. However, the role of S-layer
could operate in the gastrointestinal tract (Spencer and proteins in the adhesion of L. acidophilus to intestinal
Chesson 1994), and in our previous work, L. acidophilus epithelial cells is not clearly understood. While Green and
M92 showed antagonistic activity against enteropathogenic Klaenhammer (1994) observed that carbohydrate and pro-
E. coli and S. Typhimurium (Šušković et al. 1997). tein substances on the cell surface other than those in the
Coaggregation with potentially gut pathogens could there- S-layer are responsible for adhesion, Schneitz et al. (1993)
fore contribute to the probiotic properties ascribed to lactic stated that the opposite was true. Furthermore, Mukai and
acid bacteria. Arihara (1994) found that glycoproteins in the S-layer on the
In order to gain information on the structural properties surface of L. acidophilus JCM 1132 bind to lectins on the
of the cell surface of L. acidophilus M92 that are responsible intestinal epithelial cells.
for aggregation and adhesion, its hydrophobicity/hydrophi- In conclusion, our findings indicate that there is a
licity was compared with two other lactic acid bacteria relationship between autoaggregation and adhesiveness of
(L. plantarum L4 and Ent. faecium L3). The fact that a high L. acidophilus M92 that are mediated by proteinaceous
percentage of L. acidophilus M92 cells adhered to xylene, an components on the cell surface.
apolar solvent, demonstrated hydrophobic cell surface of
this strain. However, L. plantarum L4 and Ent. faecium L3
ACKNOWLEDGEMENTS
showed more hydrophilic cell surface properties, and strong
affinities to chloroform, which means they are strong The authors are grateful for financial support from The
electron donors. Many previous studies on the physico- Ministry of Science and Technology of Republic Croatia
chemistry of microbial cell surfaces have shown that the (Projects: Probiotic activity of lactic acid bacteria – 058403
presence of (glyco-) proteinaceous material at the cell surface and Hypocholesterolemic activity of lactic acid bacteria –
results in higher hydrophobicity, whereas hydrophilic 058001).
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 94, 981–987
 ADHESION OF L. ACIDOPHILUS M92 987

Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paraca-

REFERENCES
sei, and Lactobacillus rhamnosus strains. Applied and Environmental
Alander, M., Korpela, R., Saxelin, M., Vilpponen-Salmela, T., Microbiology 63, 1725–1731.
Matilla-Sandholm, T. and Wright, A. (1997) Recovery of Lactoba- Pérez, P.F., Minnaard, Y., Disalvo, E.A. and de Antoni, G.L. (1998)
cillus rhamnosus GG from human colonic biopsies. Letters in Applied Surface properties of bifidobacterial strains of human origin. Applied
Microbiology 24, 361–364. and Environmental Microbiology 64, 21–26.
Bellon-Fontaine, M.N., Rault, J. and van Oss, C.J. (1996) Microbial Pouwels, P.H., Leer, R.J., Shaw, M., Heijne den Bak-Glashouwer,
adhesion to solvents: a novel method to determine the electron- M.J., Tielen, F.D., Smit, E., Martinez, B., Jore, J. and Conway,
donor/electron-acceptor or Lewis acid-base properties of microbial- P.L. (1998) Lactic acid bacteria as antigen delivery vehicles for oral
cells. Colloids and Surfaces 7, 47–53. immunization purposes. International Journal of Food Microbiology
Boris, S., Suárez, J.E. and Barbés, C. (1997) Characterization of the 41, 155–167.
aggregation promoting factor from Lactobacillus gasseri, a vaginal Reid, G., McGroarty, J.A., Angotti, R. and Cook, R.L. (1988)
isolate. Journal of Applied Microbiology 83, 413–420. Lactobacillus inhibitor production against Escherichia coli and
Conway, P.L. and Kjellberg, S. (1989) Protein-mediated adhesion of coaggregation ability with uropathogens. Canadian Journal of
Lactobacillus fermentum strain 737 to mouse stomach squamous Microbiology 34, 344–351.
epithelium. Journal of General Microbiology 135, 1175–1186. Reid, G., Cuperus, P.L., Bruce, A.W., Van der Mel, H.C., Tomzek,
Crow, V.L. and Gopal, P.K. (1995) Cell surface differences of L., Khoury, A.H. and Bussches, H. J. (1992) Comparison of contact
lactococcal strains. International Dairy Journal 5, 45–68. angles and adhesion to hexadecane of urogenital, dairy, and poultry
Del Re, B., Sgorbati, B., Miglioli, M. and Palenzona, D. (2000) lactobacilli: effect of serial culture passages. Applied and Environ-
Adhesion, autoaggregation and hydrophobicity of 13 strains of mental Microbiology 58, 1549–1553.
Bifidobacterium longum. Letters in Applied Microbiology 31, 438–442. Rojas, M. and Conway, P.L. (1996) Colonization by lactobacilli of
Freter, M. (1992) Factors affecting the microecology of the gut. In piglet small intestinal mucus. Journal of Applied Bacteriology 81,
Probiotics. The Scientific Basis ed. Fuller, R. pp. 111–145. Glasgow: 474–480.
Chapman & Hall. Rosenberg, M., Gutnick, D. and Rosenberg, E. (1980) Adherence of
Green, J.D. and Klaenhammer, T.R. (1994) Factors involved in bacteria to hydrocarbons: a simple method for measuring cell-surface
adherence of lactobacilli to human Caco-2 cells. Applied and hydrophobicity. FEMS Microbiology Letters 9, 29–33.
Environmental Microbiology 60, 4487–4494. Sára, M. and Sleytr, U.B. (2000) S-layer proteins. Journal of
Handley, P.S., Harty, D.W.S., Wyatt, J.E., Brown, C.R., Doran, J.P. Bacteriology 182, 859–868.
and Gibbs, A.C.C. (1987) A comparison of the adhesion, coaggre- Schneitz, C., Nuotio, L. and Lounatma, K. (1993) Adhesion of
gation and cell-surface hydrophobicity properties of fibrilar and Lactobacillus acidophilus to avian intestinal epithelial cells mediated
fimbriate strains of Streptococcus salivarius. Journal of General by the crystalline bacterial cell surface layer (S-layer). Journal of
Microbiology 133, 3207–3217. Applied Bacteriology 74, 290–294.
Kimoto, H., Kurisaki, J., Tsuji, T.M., Ohmomo, S. and Okamoto, T. Spencer, R.J. and Chesson, A. (1994) The effect of Lactobacillus spp.
(1999) Lactococci as probiotic strains: adhesion to human entero- on the attachment of enterotoxigenic Escherichia coli to isolated
cyte-like Caco-2 cells and tolerance to low pH and bile. Letters in porcine enterocytes. Journal of Applied Bacteriology 77, 215–220.
Applied Microbiology 29, 313–316. Šušković, J., Brkić, B., Matošić, S. and Marić, V. (1997) Lactobacillus
Kos, B. (2001) Probiotic Concept: In Vitro Investigations with Chosen acidophilus M92 as potential probiotic strain. Milchwissenschaft 52,
Lactic Acid Bacteria, PhD Thesis, Faculty of Food Technology and 430–435.
Biotechnology, University of Zagreb, Croatia. Šušković, J., Kos, B., Matošić, S. and Besendorfer, V. (2000) The
Kos, B., Šušković, J., Goreta, J. and Matošić, S. (2000) Effect of effect of bile salts on survival and morphology of a potential
protectors on the viability of Lactobacillus acidophilus M92 in probiotic strain Lactobacillus acidophilus M92. World Journal of
simulated gastrointestinal conditions. Food Technology and Biotech- Microbiology and Biotechnology 16, 673–678.
nology 38, 121–128. Šušković, J., Kos, B., Goreta, J. and Matošić, S. (2001) Role of lactic
Laemmli, U.K. (1970) Clevage of structural proteins during the acid bacteria and bifidobacteria in synbiotic effect. Food Technology
assembly of the head of bacteriophage T4. Nature (London) 227, and Biotechnology 39, 227–235.
680–685. Švob, M. (1974) Histološke i histokemijske metode pp. 297–298. Sarajevo:
Mäyrä-Mäkinen, A., Manninen, M. and Gyllenberg, H. (1983) The Svjetlost.
adherence of lactic acid bacteria to the columnar epithelial cells of Tannock, G.W. (1999) A fresh look at the intestinal microflora. In
pigs and calves. Journal of Applied Microbiology 55, 241–245. Probiotics. A Critical Review ed. Tannock, G.W. pp. 5–14.
Mukai, T. and Arihara, K. (1994) Presence of intestinal lectin-binding Wymondham: Horizon Scientific Press.
glycoproteins on the cell surface of Lactobacillus acidophilus. Vandevoorde, L., Christiaens, H. and Verstraete, W. (1992) Prevalence
Bioscience Biotechnology and Biochemistry 58, 1851–1854. of coaggregation reactions among chicken lactobacilli. Journal of
Pedersen, K. and Tannock, G.W. (1989) Colonization of the porcine Applied Bacteriology 72, 214–219.
gastrointestinal tract by lactobacilli. Applied and Environmental Wadström, T., Andersson, K., Sydow, M., Axelsson, L., Lindgren, S.
Microbiology 55, 279–283. and Gullmar, B. (1987) Surface properties of lactobacilli isolated
Pelletier, C., Bouley, C., Cayuela, C., Bouttier, S., Bourlioux, P. and from the small intestine of pigs. Journal of Applied Bacteriology 62,
Bellon-Fontaine, M.N. (1997) Cell surface characteristics of 513–520.

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