JFS M: Food Microbiology and Safety

Indigenous Dadih Lactic Acid

Bacteria: Cell-Surface Properties
and Interactions with Pathogens
M. CARMEN COLLADO, INGRID SURONO, JUSSI MERILUOTO, AND SEPPO SALMINEN

ABSTRACT: Cell surface properties of dadih lactic acid bacteria strains were studied for adhesion to hydrocarbons
(BATH) and aggregation abilities. Autoaggregation correlates with adhesion, which is a prerequisite for colonization
and infection of the gastrointestinal tract by many pathogens, whereas coaggregation has been related to the ability
to interact closely with pathogens. The results demonstrated significant differences in cell surface properties among
the tested natural lactic acid bacteria food strains. Hydrophobicity increased when the cells were heat inactivated.
All strains showed aggregation abilities with the pathogen strains tested, but the coaggregation properties were
strain-specific. Our results indicate that the ability to autoaggregate, together with cell surface hydrophobicity and
coaggregation abilities with pathogen strains, can be used for preliminary screening in order to identify potentially
probiotic bacteria suitable for human or animal use. This study suggest the importance to identify and characterize
bacterial cell-wall properties to understand their role in adhesion to hydrocarbons, autoaggregation and relation to
coaggregation mechanisms, and also the relevance to future probiotic food development from natural strains.
Keywords: adhesion, coaggregation, dadih, pathogens, probiotics

Introduction Schellenberg and others 2006). This may be due to the production

T he protective role of probiotic bacteria against gastrointesti-

nal pathogens and the underlying mechanisms are unclear.
Pathogen inhibition by lactic acid bacteria may provide significant
protection against pathogens either through a natural competitive
barrier against pathogens in the gastrointestinal tract or as a method
to decontaminate bacterial toxins. The ability to adhere to mucosal
surfaces has been suggested to be an important property of many
bacterial strains used as probiotics. Adherence of bacteria to in-
testinal epithelium is required for both colonization and infection
by many pathogens (Freter 1992). Adherence of bacterial cells is re-
lated to cell surface characteristics (Bibiloni and others 2001; Canzi
and others 2005). A number of reports have described the composi-
of a microenvironment around the pathogens and generation of in-
hibitory substances by lactobacilli and other lactic acid bacteria. A
relationship between autoaggregation and adhesion ability has been
reported for some bifidobacterial species (Perez and others 1998; Del
Re and others 2000). A correlation between adhesion ability and hy-
drophobicity, as measured by microbial adhesion to hydrocarbons,
has been reported for some lactobacilli (Wadström and others 1987;
Del Re and others 2000), but also conflicting results have been re-
ported (Vinderola and others 2004).
Dadih is Indonesian traditional fermented buffalo milk and is
believed to be beneficial for human health (Akuzawa and Surono
2002). The competitive exclusion of contaminants and pathogens

M: Food Microbiology and Safety

tion, structure, and forces of interaction related to bacterial adhesion
(Pelletier and others 1997; Pérez and others 1998; Del Re and oth-
ers 2000; Tuomola and others 2000; Collado and others 2005, 2006).
The results are sometimes difficult to interpret, but in many cases,
aggregation ability is related to cell adherence properties (Vande-
voorde and others 1992; Boris and others 1997; Del Re and others
2000). Bacterial aggregation between microorganisms of the same
strain (autoaggregation) or between different species and strains
(coaggregation) is of considerable importance in several ecological
niches, especially in the human gut where probiotics are to be ac-
tive (Jankovic and others 2003). Several authors have reported that
the coaggregation abilities of Lactobacillus species may enable it
to form a barrier to prevent colonization by pathogens (Reid and
others 1988; Boris and others 1997; Schachtsiek and others 2004;
MS 20060392 Submitted 7/17/2006, Accepted 1/11/2007. Authors Collado,
Surono, and Salminen are with Functional Foods Forum, Univ. of Turku,
Itäinen Pitkäkatu 4A, FI-20014 Turku, Finland. Author Surono is with
Seameo-Tropmed RCCN Univ. of Indonesia, Salemba Raya 6 Jakarta 10430,
Indonesia. Author Meriluoto is with Dept. of Biochemistry and Pharmacy,
Åbo Akademi Univ., Tykistökatu 6A, FI-20520 Turku, Finland. Direct in-
quiries to author Collado (E-mail: marcol@utu.fi).
could be due to the presence of the natural indigenous lactic acid
bacteria derived from bamboo tubes, buffalo milk, or banana leaves
involved in milk fermentation. Some studies on probiotic properties
of indigenous strains isolated from dadih fermented milk demon-
strated to exhibit antimutagenic, acid, and bile tolerance as well as
antipathogenic properties (Surono and Hosono 1996; Surono 2003).
The objective of this study was to determine cell surface char-
acteristics (hydrophobicity, autoaggregation, and coaggregation
abilities) of lactic acid bacteria strains isolated from dadih, tra-
ditional fermented milk product from Indonesia, with selected
enteric pathogens. Selection of new natural probiotics that inhibit
or displace a specific pathogen can be based on further assessment,
product development, and human clinical interventions on preven-
tion or treatment of infection caused by the pathogen. This would
enhance human health and have a positive economic impact, espe-
cially in developing countries.
Materials and Methods
Bacterial strains
The indigenous dadih LAB strains were Lactobacillus plantarum
strains IS-10506 and IS-20506 and Enterococcus faecium strain


C 2007 Institute of Food Technologists Vol. 72, Nr. 3, 2007—JOURNAL OF FOOD SCIENCE M89
doi: 10.1111/j.1750-3841.2007.00294.x
Further reproduction without permission is prohibited
 Properties of dadih isolates and interactions with pathogens. . .

IS-27526, IS-23427 and IS-16183. These strains were cultured in MRS
broth (Scharlau Microbiology, Barcelona, Spain) for 48 h at 37 ◦ C,
harvested by centrifugation, and freeze-dried, and they were stored
in Functional Foods Forum Culture Collection, University of Turku,
Finland. For assays with freshly grown bacteria, the bacteria were
cultured in MRS broth for 18 h at 37 ◦ C under aerobic conditions,
harvested by centrifugation (3200 × g, 4 ◦ C, 20 min), and washed
twice with phosphate-buffered saline buffer (PBS: 130 mM sodium
chloride, 10 mM sodium phosphate, pH 7.2). The viability of the cells
was tested in MRS agar plates.
The bacterial pathogens used in this study were selected for their
relevance as established on emerging foodborne pathogens, and
they were Bacteroides vulgatus DSM 1447, Clostridium histolyticum
DSM 627, Clostridium difficile DSM 1296, Escherichia coli K2, En-
terobacter sakazakii ATCC 29544, and Staphylococcus aureus DSM
20231. Pathogens were grown in Gifu anaerobic medium (GAM Nis-
sui Pharmaceutical, Tokyo, Japan) under anaerobic conditions (10%
H 2 , 10% CO 2 , and 80% N 2 ; Concept 400 anaerobic chamber, Ruskinn
Technology, Leeds, UK) for 18 h at 37 ◦ C.
Statistical analysis
The results for the adhesion assay were expressed as the average
of 3 or 4 independent experiments. Each experiment was performed
with 4 parallels to correct for intra-assay variation. Statistical analy-
sis was made by SPSS 11.0 software (SPSS Inc., Chicago, Ill., U.S.A.).
Data were subjected to a 1-way analysis of variance.
Results and Discussion
T he abilities to adhere to mucosal surfaces have been suggested
to be important properties of bacterial strains used as probi-
otics. In addition, bacterial aggregation is of considerable impor-
tance in several ecological niches, especially in the human gut where
probiotics are to be active (Jankovic and others 2003). Thus, the ob-
jective of this work was to study the relationship among cell surface
properties such as hydrophobicity, aggregation abilities, and the ad-
hesion of potentially probiotic strains. The overall objective was to
assess whether bacterial properties such as hydrophobicity and ag-
gregation ability could be “predictive” and detect the most useful
probiotic strains for different targets.

Bacterial adhesion to hydrocarbons

Bacterial adhesion to hydrocarbons
The bacterial adhesion to hydrocarbons (BATH) test was per-
formed according to Rosenberg and others (1980). Briefly, bacterial
cells were washed with PBS buffer and resuspended in the same
buffer. Absorbance was adjusted to 0.25 ± 0.05 to standardize the
number of bacteria (108 cells/mL) at 600 nm. Then, equal propor-
tions of viable bacterial suspension and solvent (xylene) were mixed
by vortexing for 5 min. The 2-phase system appeared and the aque-
ous phase was removed after 1 h of incubation at room temperature
and its absorbance was measured. Also, we analyzed the viability
influence in the BATH test by means of mixing heat inactivated bac-
terial suspensions (98 ◦ C during 10 min) with xylene. Results were
reported as a percentages from 3 replicates according to the formula
BATH% = [(A0 – A)/A0] × 100, where A0 and A are absorbance before
and after mixing with xylene, respectively.
Bacterial autoaggregation analysis
Aggregation abilities of dadih isolates were screened by the visual
and spectophotometry assays. Briefly, 108 cells/mL of overnight cul-
ture were harvested by centrifugation at 3200 × g and 4 ◦ C for 20 min,
M: Food Microbiology and Safety
The BATH test has been extensively used for measuring cell sur-
face hydrophobicity in lactic acid bacteria (Pérez and others 1998;
Kos and others 2003; Vinderola and others 2004; Canzi and others
2005). The high percentage of some cells adhering to xylene, a apo-
lar solvent, demonstrated hydrophobic cell surface properties. The
adhesion percentages of lactic acid bacteria isolated from dadih fer-
mented milk and pathogen strains to the tested hydrocarbons are
shown in Table 1. The results indicate that, in general, after heat
treatment the hydrophobicity percentages increased 9.6% to 17.8%
for all lactic acid bacteria and 3.0% to 53.8% for pathogens. Excep-
tions such as Clostridium difficile DSM 1296 show a reduction in
percentage hydrophobicity after heat treatment (P > 0.05).
The most hydrophophic viable pathogens strains were B. vulga-
tus (51.4%), C. difficile (72.6%), and Staphylococcus aureus strain
(54.6%). The percentages for heat-treated cells were from 75.6% to
57.6%, respectively, for the same strains and for C. histolyticum were
61.6%, E. coli, 70.1%, and Enterobacter sakazakii, 78.4%. In general,
Table 1 --- Adhesion to hydrocarbons of viable and nonvi-
able probiotic and pathogenic strains as measured using
the BATH test. Results are expressed as a mean ± stan-

washed 3 times with PBS buffer, and suspended in the same buffer.
dard deviation (SD) (n ≥ 4).
The mixture was vortexed and then incubated at 20 ◦ C and 37 ◦ C
for 24 h. Spectophotometry assay was determined as described by Percentages of adhesion
to hydrocarbons
Reniero and others (1992) as the aggregation percentage expressed
as 1 – (A Time /A initial ) × 100 where A Time represents the absorbance Live Nonviable
Pathogen strains (mean ± SD) (mean ± SD)
of the mix at different times (0, 2, 16, 20, 24 h), measured at 600 nm.
Bacteroides vulgatus DSM 1447 51.4 ± 13.9∗ 75.6 ± 13.6
Clostridium histolyticum DSM 627 32.2 ± 1.8∗ 61.6 ± 4.6
Bacterial coaggregation analysis Clostridium difficile DSM 1296 72.6 ± 2.8 68.9 ± 15.2
The coaggregation analysis was performed according to Hand- Escherichia coli K2 32.7 ± 14.5∗ 70.1 ± 13.8
Enterobacter sakazakii ATCC 29544 24.6 ± 19.7∗ 78.4 ± 17.5
ley and others (1987). Briefly, bacterial suspensions were prepared Staphylococcus aureus DSM 20231 54.6 ± 15.8 57.7 ± 11.34
as described above. Equal volumes of cells of the different probi-
Percentages of adhesion
otic and pathogen strains (1:1 v/v) were mixed and incubated at
to hydrocarbons
20 and 37 ◦ C without agitation. Absorbances (A 600 ) of the mixtures
described above were monitored during 2 h of incubation. Percent- Live Nonviable
Probiotic strains (mean ± SD) (mean ± SD)
ages of coaggregation were determined as [(A patho + A probio )/2 −
(A mix )/(A patho + A probio )/2] × 100, where A patho and A probio repre- Enterococcus faecium IS-16183 25.1 ± 8.8 34.7 ± 12.3
Enterococcus faecium IS-23427 46.3 ± 12.0 55.9 ± 20.3
sent the absorbance at 600 nm in control tubes containing only the
Enterococcus faecium IS-27526 63.9 ± 10.0∗ 81.7 ± 12.0
pathogen or dadih strain, respectively, after 2 h of incubation and Lactobacillus plantarum IS-10506 76.3 ± 16.7 87.6 ± 14.8
A mix represents the absorbance of the mixed culture after the same Lactobacillus plantarum IS-20506 10.3 ± 6.3 20.2 ± 8.4
period of incubation. The standard deviations were derived from ∗
Significant differences (P < 0.05) between hydrophobicity percentages
the coaggregation values of 3 independent experiments. achieved with live and dead cells.

M90 JOURNAL OF FOOD SCIENCE—Vol. 72, Nr. 3, 2007

Properties of dadih isolates and interactions with pathogens. . .

Table 2 --- Percentages of autoaggregation for pathogen and probiotic strains from dadih strains at different times
(control time = 0 h)
Percentages of autoaggregation Percentages of autoaggregation
(20 ◦ C) (37 ◦ C)
2h 16 h 20 h 24 h 2h 16 h 20 h 24 h
Pathogen strains
Bacteroides vulgatus DSM 1447 2.7 ± 0.2 11.5 ± 2.3 12.7 ± 2.5 31.5 ± 10.0 1.8 ± 0.3 5.7 ± 2.2 11.6 ± 3.5 27.5 ± 5.0
Clostridium histolyticum DSM 627 0.0 ± 0.5 30.7 ± 8.8∗ 31.7 ± 5.8∗ 63.1 ± 6.9∗ 0.2 ± 0.5 3.0 ± 1.0 30.6 ± 2.5∗ 58.6 ± 6.2∗
Clostridium difficile DSM 1296 8.2 ± 1.3 8.6 ± 1.2 9.7 ± 3.5 48.1 ± 7.0 7.1 ± 2.2 11.2 ± 2.5 11.6 ± 5.5 43.9 ± 5.0
Escherichia coli K2 2.5 ± 0.6 2.5 ± 1.0 3.2 ± 1.1 43.6 ± 4.4 2.8 ± 0.5 5.5 ± 2.2 6.1 ± 3.5 39.6 ± 3.8
Enterobacter sakazakii ATCC 29544 3.7 ± 1.4 4.9 ± 1.5 6.6 ± 0.8 41.0 ± 3.6 2.5 ± 1.1 6.7 ± 3.0 7.5 ± 3.2 37.0 ± 6.0
Staphylococcus aureus DSM 20231 28.3 ± 6.1∗ 28.7 ± 1.0∗ 29.4 ± 3.3∗ 62.4 ± 5.0∗ 11.3 ± 0.5∗ 31.3 ± 3.7∗ 38.3 ± 5.5∗ 58.4 ± 6.0∗
Probiotic strains
Enterococcus faecium IS-16183 12.1 ± 4.2 13.8 ± 2.0 17.4 ± 5.0 37.7 ± 6.8 10.0 ± 2.6 14.8 ± 4.5 38.6 ± 5.5 38.6 ± 9.0
Enterococcus faecium IS-23427 11.6 ± 5.0 13.7 ± 4.0 16.7 ± 2.3 32.3 ± 5.5 5.9 ± 1.6 36.2 ± 9.0 54.6 ± 10.0 42.4 ± 8.0
Enterococcus faecium IS-27526 0.6 ± 0.5∗ 1.4 ± 0.5∗ 14.2 ± 2.5 20.6 ± 4.2 8.5 ± 3.0 13.8 ± 5.0 34.1 ± 8.0 37.8 ± 5.5
Lactobacillus plantarum IS-10506 10.1 ± 2.5 12.1 ± 2.3 13.5 ± 3.0 32.8 ± 7.0 9.0 ± 2.0 13.8 ± 3.0 25.5 ± 3.9 34.8 ± 6.3
Lactobacillus plantarum IS-20506 1.6 ± 0.5∗ 3.8 ± 2.0∗ 7.2 ± 2.4∗ 26.7 ± 5.0 0.0 ± 0.5∗ 22.8 ± 5.7 41.6 ± 9.0 58.4 ± 10.0
∗
Significant differences among all strains tested in each time.

LAB strains tested showed a lower hydrophobicity than pathogens. IS-20506 and E. faecium IS-23427 with more than 45% of autoag-
The strains with lowest hydrophobicity were L. plantarum IS-20506 gregation, while the worst strain was L. plantarum IS-10506 with
and Enterococcus faecium IS-16183, 10.3% and 25.1%, respectively, 25.5%.
for viable cells and 20.2% and 34.7%, respectively, for heat-treated By plotting percentages of autoaggregation vs. BATH values (Fig-
cells. ure 1) at 20 ◦ C, it has been possible to verify that a wide range of di-
versity exists in cell surface properties among pathogens and probi-
Bacterial autoaggregation analysis
otics. BATH values were related to autoaggregation properties (Fig-
The autoaggregation properties of pathogens and probiotic
ure 1) because strains with higher adhesion to hydrocarbons showed
strains tested were characterized over a period of 24 h by visual
high autoaggregation abilities. L. plantarum IS-10506 and E. faecium
observation of aggregates formed. In general, the probiotic strains
IS-23427 and IS-27526 presented the highest percentages of adhe-
presented higher autoaggregation abilities than the pathogens at
sion to hydrocarbons and also, highest autoaggregation abilities,
both temperatures tested (20 and 37 ◦ C). These temperatures were
and they appear in the right part in the graph (Figure 1). L. plan-
chosen because 20 ◦ C takes the dadih fermentation and 37 ◦ C is
tarum IS-20506 strain showed a low percentage of adhesion to hy-
the natural body temperature. The results obtained by visual score
drocarbon as well as a low autoaggregation ability. Different authors
and absorbance measurements were correlated (data not shown),
had suggested that the bacterial aggregation could be the factor in
and absorbance measurements are shown in Table 2. The most au-
promoting the gut colonization of beneficial microorganisms (Ce-
toaggregative strains among pathogens tested were St. aureus DSM
sena and others 2001; Jankovic and others 2003). In addition, these
20231 and C. histolyticum DSM 627 at both temperatures tested (20
and 37 ◦ C) and all times analyzed (2, 16, 20, and 24 h). In general,
all strains isolated from Indonesian fermented milk product show
higher percentages of autoaggregation from 7.2% to 17.4% after 20
h at 20 ◦ C and 25.5% to 54.6% after 20 h at 37 ◦ C and also, these per-
centages after 24 h of incubation were 20.6% to 37.7% at 20 ◦ C and
34.8% to 58.4% at 37 ◦ C. Autoaggregation percentages of pathogen

M: Food Microbiology and Safety

strains were not significantly different (P > 0.05) at 20 and 37 ◦ C
in all times tested whereas for probiotic strains, autoaggregation
percentages were significant different (P < 0.05) between 20 and
37 ◦ C from 20 to 24 h.
Interestingly, after 2 h incubation the strain with highest autoag-
gregation abilities was St. aureus DSM 20231 for pathogen strains
and L. plantarum IS-10506 and Enterococcus faecium IS-16183 and
IS-23427 for dadih strains. These dadih strains, L. plantarum IS-
10506 and Enterococcus faecium IS-16183 and IS-23427, showed the
highest aggregation abilities among all the dadih strains tested. In
general, no differences within a strain were observed between incu-
bation temperatures at 20 and 37 ◦ C.
The most autoaggregative pathogen strains were C. histolyticum
DSM 627 and St. aureus DSM 20231, 31.7% and 29.4% respectively,
at 20 ◦ C, whereas at 37 ◦ C were 30.6% and 38.3%, respectively, after
20 h incubation. These results were also confirmed by visual scores.
The strains with the best autoaggregation abilities among probiotics Figure 1 --- Autoaggregation abilities (%) of the probiotic
strains tested, after 20 h incubation at 20 ◦ C, were E. faecium IS-23427 bacteria isolated from dadih as a function of adhesion
and IS-16183 strains (>16.0%) while the least autoaggregative strain of xylene (%) after incubation at room temperature (20
◦

( r) lower aggregation and adhesion, and (
) higher aggre-

C). Values are the average from at least 3 experiments.
was L. plantarum IS-20506 (7.2%) under the same circumstances.
The best strains after 20 h incubation at 37 ◦ C were L. plantarum gation and variable adhesion (between 13% and 18%).

Vol. 72, Nr. 3, 2007—JOURNAL OF FOOD SCIENCE M91

 Properties of dadih isolates and interactions with pathogens. . .

results are supported by previous studies with commercial probiotic demonstrated to be strain-specific and depend on time and incu-
bacteria (Collado and others in press). bation conditions. Moreover, the best abilities to coaggregate with
pathogens were shown by L. plantarum IS-20506 dadih strain and
Bacterial coaggregation analysis the worst abilities by E. faecium IS-27526.
To quantify the cell adherence, a coaggregation assay was devel- Thus, the results are in agreement with the hypothesis that co-
oped, which established coaggregation between probiotic strains aggregation abilities are related with autoaggregation properties
and the potential foodborne pathogens B. vulgatus, C. histolyticum and strains with higher autoaggregation showed high coaggrega-
and difficile, St. aureus, Enterobacter sakazakii, and E. coli. Coaggre- tion abilities among pathogens tested. A general correlation be-
gation with potential gut pathogens could therefore contribute to tween bacterial overall surface characteristics (adhesion to hydro-
the probiotic properties ascribed to specific lactic acid bacteria. All carbons) and binding capabilities (autoaggregation and coaggrega-
the strains isolated from dadih fermented milk product showed co- tion) is possible, but the strains have to be assessed on a case-by-case
aggregation abilities with the pathogens tested, but the percentages basis importance of natural strains for probiotic food development.
depended on each strain (probiotic and pathogen strains) and also Our findings indicate that the ability to autoaggregate, together with
time of co-incubation. The coaggregation properties were analyzed cell surface hydrophobicity as measured by BATH and coaggrega-
at 20 and 37 ◦ C and they were not significantly (P > 0.05) different; tion abilities with pathogen strains, could be used for preliminary
hence, only the data at 20 ◦ C are shown. The results of coaggre- screening to identify potentially probiotic bacteria suitable for hu-
gation were dadih strain-, pathogen strain- and time-dependent. man or animal use.
Coaggregation analysis were tested using different indicator strains
such as C. histolyticum DSM 627, B. vulgatus DSM 1447, St. au- Conclusions
reus DSM 20231 and Enterobacter sakazakii ATCC 29544, C. difficile
DSM 1296, and E. coli K2 at different times. Results are shown in Fig- A general correlation between bacterial overall surface charac-
teristics (adhesion to hydrocarbons) and binding capabili-
ure 2. All dadih strains tested showed aggregation abilities with the ties (autoaggregation and coaggregation) exists but there is vari-
pathogen strains tested, but the percentage of coaggregation was ation in the properties of specific strains and each potential

Figure 2 --- Coaggregation abilities of

probiotic strains with pathogens
after 2h incubation at 20 ◦ C
expressed as percentages. Values
are the average ± SD from at least 3
experiments.
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M92 JOURNAL OF FOOD SCIENCE—Vol. 72, Nr. 3, 2007

Properties of dadih isolates and interactions with pathogens. . .
probiotic needs to be independently tested. It is important to iden-
tify and characterize bacterial cell-wall properties to understand
their role in adhesion to hydrocarbons, autoaggregation, and re-
lation to coaggregation mechanisms, and also a relevance to fu-
ture probiotic food development from natural strains that would
enhance human health and also have a positive economic impact
especially in developing countries.
Acknowledgments
This work was supported by the Academy of Finland, Research
Council for Biosciences and Environment (decision numbers
210309 to Åbo Akademi and 210310 to Univ. of Turku). M.C. Col-
lado is the recipient of Excellence Postdoctoral grant from Consel-
leria Empresa, Univ. y Ciencia de la Generalitat Valenciana, Spain
(BPOSTDOC 06/016).
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