ORIGINAL ARTICLE
In vitro assessment of safety and probiotic potential
characteristics of Lactobacillus strains isolated from water
buffalo mozzarella cheese
Sabrina Neves Casarotti
1,2
& Bruno Moreira Carneiro
3
& Svetoslav Dimitrov Todorov
4
&
Luis Augusto Nero
4
& Paula Rahal
5
& Ana Lúcia Barretto Penna
1
Received: 10 August 2016 /Accepted: 7 February 2017 /Published online: 28 February 2017
#
Springer-Verlag Berlin Heidelberg and the University of Milan 2017
Abstract The aim of this study was to evaluate the safety and
probiotic potential characteristics of ten Lactobacillus spp.
strains (Lactobacillus fermentum SJRP30, Lactobacillus casei
SJRP37, SJRP66, SJRP141, SJRP145, SJRP146, and
SJRP169, and Lactobacillus delbrueckii subsp. bulgaricus
SJRP50, SJRP76, and SJRP149) that had previously been
isolated from water buffalo mozzarella cheese. The safety of
the strains was analyzed based on mucin degradation, hemo-
lytic activity, resistance to antibiotics and the pre sence of
genes encoding virulence factors. The in vitro tests concerning
probiotic potential included survival under simulated gastro-
intestinal (GI) tract conditions, intestinal epithelial cell adhe-
sion, the presence of genes encoding adhesion, aggregation
and colonization factors, antimicrobial activity, and the pro-
duction of the β-galactosidase enzyme. Although all strains
presented resistance to several antibiotics, the resistance was
limited to antibiotics to which the strains had intrinsic resis-
tance. Furthermore, the strains presented a limited spread of
genes encoding virulence factors and resistance to antibiotics,
and none of the strains presented hemolytic or mucin degra-
dation activity. The L. delbrueckii subsp. bulgaricus strains
showed the lowest survival rate after exposure to simulated GI
tract conditions, whereas all of the L. casei and L. fermentum
strains showed good survivability. None of the tested lactobacilli
strains presented bile salt hydrolase (BSH) activity, and only
L. casei SJRP145 did not produce the β-galactosidase enzyme.
The strains showed varied levels of adhesion to Caco-2 cells.
None of the cell-free supernatants inhibited the growth of path-
ogenic target microorganisms. Overall, L. fermentum SJRP30
and L. casei SJRP145 and SJRP146 were revealed to be safe
and to possess similar or superior probiotic characteristics com-
pared to the reference strain L. rhamnosus GG (ATCC 53103).
Keywords Lactic acid bacteria
.
Dairy
.
Safety
.
Antibiotic
resistance
.
Beneficial properties
.
Gastrointestinal tract
survival
Introduction
Lactobacillus spp. belong to the group of lactic acid bacteria
(LAB) and have a long history of use in the production of
dairy products due to their ability to convert lactose into lactic
acid (Tulumoğlu et al. 2014). In addition to their use as tech-
nological agents in the food industry, some Lactobacillus spe-
cies can confer health benefits to the host when they are ad-
ministered adequately as probiotics. Probiotics are currently
defined Bas live microorganisms that, when administered in
adequate amounts, confer health benefit on the host^ (Hill
et al. 2014). Althou gh probiotics have been extensively
* Sabrina Neves Casarotti
sabrinacasarotti@yahoo.com.br
* Ana Lúcia Barretto Penna
analucia@ibilce.unesp.br
1
Departamento de Engenharia e Tecnologia de Alimentos, Instituto de
Biociências, Letras e Ciências Exatas, Universidade Estadual
Paulista (UNESP), R. Cristovão Colombo, 2265, 15054-000 São
José do Rio Preto, São Paulo, Brazil
2
Departamento de Alimentos e Nutrição, Faculdade de Nutrição,
Universidade Federal de Mato Grosso (UFMT),
78060-900 Cuiabá, MT, Brazil
3
Instituto de Ciências Exatas e Naturais, Universidade Federal de
Mato Grosso (UFMT), 78735-901 Rondonópolis, MT, Brazil
4
Departamento de Veterinária, Universidade Federal de Viçosa
(UFV), 36570-000 Viçosa, MG, Brazil
5
Departamento de Biologia, Instituto de Biociências, Letras e Ciências
Exatas, Universidade Estadual Paulista (UNESP), Rua Cristóvão
Colombo, 2265, 15054-000 São José do Rio Preto, SP, Brazil
Ann Microbiol (2017) 67:289–301
DOI 10.1007/s13213-017-1258-2
studied and commercialized, and are the subject of national
and international regulations, there is no agreement
concerning the amount of probiotic bacteria necessary to pro-
duce their beneficial effects. Generally, probiotic food prod-
ucts must contain 10
6
CFU/mL or CFU/g (Shah 2000).
Nevertheless, some authors state that beneficial effects can
be achieved even when bacteria lose their viability (Adams
2010).
Some of the health effects attributed to probiotic consump-
tion include the regulation of gastrointestinal (GI) functions,
relief of lactose intolerance, prevention of different types of
diarrhea besides urogenital infections, reduction in cholesterol
levels, reduction in atopic and food allergies, and modulation
of the immune system. Furthermore, in vitro studies have
shown that probiotic bacteria reduce the number of pathogens
and their metabolic activities in the human intestine and com-
pete with these microorganisms for attachment sites to intes-
tinal epithelial cells and nutrients (Guarner and Malagelada
2003;Mishraetal.2015).
Although a large number of probiotic strains are available
for commercial use worldwide, the isolation and characteriza-
tion of new strains from different species is desirable; thus,
many studies in this field have been published in recent years
(Jeronymo-Ceneviva et al. 2014; Peres et al. 2014; de Paula
et al. 2015; Oh and Jung 2015). Probiotics targeted for human
consumption are usually isolated from humans or animals
because strains from these origins can better adapt to the con-
ditions encountered in the human/animal GI tract, which en-
ables more successful gut colonization (Argyri et al. 2013).
However, certain food-associated Lactobacillus strains have
probiotic characteristics even though they do not belong to the
gut microbiota (Solieri et al. 2014; Tulumoğlu et al. 2014).
According to the FAO/WHO (2002), a bacterial strain
should fulfill a number of requirements to be considered pro-
biotic; these requirements must be verified by in vitro and
in vivo tests. In vitro tests are useful for the selection of strains
that have greater probiotic potential; these tests increase
knowledge regarding the strain as well as the mechanisms
underlying the beneficial effects. Although LAB, particularly
Lactobacillus, are generally recognized as safe (GRAS), ad-
ditional tests should be performed to check the safety of these
strains because some cases recently associated systemic infec-
tion with the consumption of probiotics (Liong 2008; Sharma
and Devi 2014). Thus, evaluating their safety, assessing their
resistance to antibiotics, investigating the presence of viru-
lence genes, and determining hemolytic activity are important
(Jeronymo-Ceneviva et al. 2014; Vijayakumar et al. 2015).
Given these points, the aim of this study was to characterize
the safety features and probiotic potential attributes of autoch-
thonous Lactobacillus spp. isolated from water buffalo moz-
zarella cheese using in vitro tests. Candidates that met the
established criteria may be used in the produ ction of
fermented products to promote their probiotic characteristics.
Materials and methods
Bacterial strains
Ten Lactobacillus strains previously isolated and identified
through 16S rRNA ge ne sequenc ing by our group (Silva
et al. 2015; Silva 2015)asLactobacillus fermentum
(SJRP30), Lactobacillus casei (SJRP37, SJRP66, SJRP141,
SJRP145, SJRP146, and SJRP169), and Lactobacillus
delbrueckii subsp. bulgaricus (SJRP50, SJRP76 and
SJRP149) were screened for their safety and probiotic poten-
tial. Lactobacillus rhamnosus GG (ATCC 53103) was used as
a probiotic reference strain. The strains were maintained at
−80 °C in MRS broth (Difco, Becton Dickinson, Sparks,
MD) supplemented with 25% (v/v) glycerol (Vetec, Duque
de Caxias, RJ, Brazil). Each culture was sub-cultured at least
twice in MRS broth before use in the assays.
Assessment of safety characteristics
Hemolytic activity
Fresh lactobacilli broth cultures (8.0–9.0 log CFU/mL) were
streaked in triplicate on Columbia agar plates containing 5%
(w/v) sheep blood (NewProv, Pinhais, PR, Brazil). After 48 h
of incubation at 37 °C, the plates were examined for hemolytic
reactions. The Lactobacillus rhamnosus GG (ATCC 53103)
and Staphylococcus aureus ATCC 6538 strains were used as
the negative and positive controls, respectively (Pieniz et al.
2014). The assay was repeated on three independent occasions
in triplicate.
Mucin degradation
Mucin degradation was determined according to Zhou et al.
(2001). Salmonella enterica subsp. enterica serovar
Typhimurium ATCC 14028 and Lactobacillus rhamnosus
GG (ATCC 53103) were used as the positive and negative
controls, respectively. The assay was repeated on three inde-
pendent occasions in triplicate.
Presence of genes encoding virulence factors, antibiotic
resistance and biogenic amines
The Lactobacillus strains were tested for the presence of vir-
ulence, antibiotic resistance and amino acid decarboxylase
genes (Table 1). DNA was extracted usin g the QIAg en
DNeasy Blood & Tissue Kit (Qiagen, H ilden, Germany),
followed by DNA concentration estimation using the
NanoDrop2000 spectrophotometer (Thermo Scientific,
Waltham, MA). PCRs were performed according to the refer-
ences listed in Table 1, and the amplified products were sep-
arated by electrophoresis in 0.8 to 2.0% (w/v) agarose gels in
290 Ann Microbiol (2017) 67:289–301
Table 1 Presence of genes implicated in virulence factors, antibiotic resistance and biogenic amine production in Lactobacillus spp. strains
Gene Encoded factor L. fermentum L. casei L. delbrueckii subsp. bulgaricus Reference
SJRP30 SJRP37 SJRP66 SJRP141 SJRP145 SJRP146 SJRP169 SJRP50 SJRP76 SJRP149
Virulence
gelE Gelatinase –
a
– + –––––+ + Vankerckhoven et al. (2004)
hyl Hyaluronidase –––––––++– Vankerckhoven et al. (2004)
asa1 Aggregation substance –––––––+ –– Vankerckhoven et al. (2004)
esp Enterococcal surface protein – +++ ––+ ––– Vankerckhoven et al. (2004)
cylA Cytolysin ––––––+ ––– Vankerckhoven et al. (2004)
efaA Endocarditis antigen – ––––––––– Martín-Platero et al. (2009)
ace Adhesion of collagen – ––––––––– Martín-Platero et al. (2009)
fsrA Gelatinase – +++ + – + ––– Lopes et al. (2006)
fsrB Gelatinase – +++ ––+ ––– Lopes et al. (2006)
fsrC Gelatinase –––––––++– Lopes et al. (2006)
sprE Serine protease ––––+ ––––+ Lopes et al. (2006)
ccf Sex pheromones, chemotactic for
human leukocytes; facilitate
conjugation
– + – +++– + –– Eaton and Gasson (2001)
cob Sex
pheromones, chemotactic for
human leukocytes; facilitate
conjugation
–––––––+ – + Eaton and Gasson (2001)
cpd Sex pheromones, chemotactic for
human leukocytes; facilitate
conjugation
– + – + – +++–– Eaton and Gasson (2001)
Biogenic amine
hdc1 Histidine decarboxylase – + –– – – + ––+deLasRivasetal.(2005)
hdc2 Histidine decarboxylase – ––––––––– de Las Rivas et al. (2005)
tdc Tyrosine decarboxylase –––––––++– de Las Rivas et al. (2005)
odc Ornithine decarboxylase – ––––––––– de Las Rivas et al. (2005)
Antibiotic resitance
van A Vancomycin resistance – +++ ––+ ––– Martín-Platero et al. (2009)
van B Vancomycin resistance – +++ – ++––– Martín-Platero et al. (2009)
vanC1 Vancomycin resistance –––+ –––+ + + Dutka-Malen et al. (1995)
vanC-2,vanC-3 Vancomycin resistance + ––– – – + ––– Dutka-Malen et al. (1995)
vanC1 Vancomycin resistance + ––– – – + ––– Miele et al. (1995)
tet
(M) Tetracycline resistance – –––––––+ – Aarestrup et al. (2000b)
tet(L) Tetracycline resistance –––+ –––++– Aarestrup et al. (2000b)
Ann Microbiol (2017) 67:289–301 291
Table 1 (continued)
Gene Encoded factor L. fermentum L. casei L. delbrueckii subsp. bulgaricus Reference
SJRP30 SJRP37 SJRP66 SJRP141 SJRP145 SJRP146 SJRP169 SJRP50 SJRP76 SJRP149
tet(K) Tetracycline resistance – + –– – – – + – + Aarestrup et al. (2000b)
tet(O) Tetracycline resistance – ++++++––– Aarestrup et al. (2000b)
tet(S) Tetracycline – ––––––––– Aarestrup et al. (2000a)
bcr(B) Bacitracin resistance ––+ – +++––– Manson et al. (2004)
bcr(D) Bacitracin resistance – ––––––––– Manson et al. (2004)
bcr(R) Bacitracin resistance –––––+ –––– Manson et al. (2004)
erm(A) Erythromycin resistance – ––––––––– Sutcliffe et al. (1996)
erm(B) Erythromycin resistance – ––––––––– Sutcliffe et al. (1996)
erm(C) Erythromycin resistance – + – ++++– + – Sutcliffe et al. (1996)
erm(B) Erythromycin resistance – ––––––––– Gevers et al. (2003)
ant(4′)-Ia Aminoglycoside resistance + + + + + – + ––– Fortina et al. (2008)
aph(3′)-II I- a Aminoglycoside resistance –
+ ––
– – + + + + Fortina et al. (2008)
aph(2″)-Ib Aminoglycoside resistance ++– +++–––– Fortina et al. (2008)
aph(2″)-Ic Aminoglycoside resistance – +++ + – + ––– Fortina et al. (2008)
aph(2″)-Id Aminoglycoside resistance –––++––+ + + Fortina et al. (2008)
aac(6′)-Ie-ap-
h(2″)-Ia
Aminoglycoside resistance + + + – + ––+ – + Fortina et al. (2008)
aac(6′)-Ii Aminoglycoside resistance + + + + + – +++– Costa et al. (1993)
catA(PIP501) Chloramphenicol resistance – ++––––––– Aarestrup et al. (2000a)
int-Tn Tetracycline resistance ––+ ––––––+ Fortina et al. (2008)
int T ranspos on integrase gene + + + + – + – +++ Geversetal.(2003)
a
+ Indicates the presence and – absence of genes
292 Ann Microbiol (2017) 67:289–301
0.5× TAE buffer. The gels were stained in 0.5× TAE buffer
containing 0.5 μg/mL of ethidium bromide (Sigma-Aldrich,
St. Louis, MO).
Antibiotic susceptibility
The disc diffusion assay was applied to determine the antibi-
otic susceptibility of the strains. Diluted culture (100 μL; 6.0
log CFU/mL) was spread onto MRS agar media (Difco), and
antibiotic discs (Oxoid, Basingstoke, UK) containing (per
disc) ampicillin (10 μg), vancomycin (30 μg), gentamicin
(10 μg), kanamycin (30 μg), streptomycin (300 μg), tetracy-
cline (30 μg), chloramphenicol (30 μg), erythr omycin
(15 μg), and clindamycin (2 μg) were placed manually on
the surface of the inoculated plates using sterile forceps.
These antibiotics were chosen according to the list proposed
by the European Food Safety Authority (EFSA 2012). The
plates were incubated at 37 °C under anaerobic conditions,
and the diameters o f the inh ibi tion zone s were ev al uate d
24 h after incubation. The susceptibility of the isolates was
scored as resistant, moderately susceptible, or susceptible ac-
cording to the cut-off values proposed by Charteris et al.
(1998). The assay was repeated on three independent occa-
sions in triplicate.
Assessment of probiotic potential characteristics
Tolerance to simulated GI tract conditions
The tolerance to simulated GI tract conditions test was per-
formed by successively exposing the strains to gastric and
enteric simulated juices as described by Botta et al. (2014).
The lactobacilli strains were grown for 18 h at 37 °C in MRS
broth, and 1 mL of each culture (8.0–9.0 CFU/mL) was dis-
tributed into four sterile flasks (two for the gastric phase and
two for the enteric phase). The solutions simulating the gastric
and enteric juices were prepared according to the method of
Bautista-Gallego et al. (2013 ). The pH values used in the
gastric and enteric phases were 2.5 and 8.0, respectively. All
enzyme solutions were prepared and filter-sterilized using a
0.22-μm membrane filter (Merck Millipore, Cork, Ireland) on
the day of analysis.
The cells were counted at the beginning (T
0
)andtheend
of t he gastric phase (T
120
) and after the enteric phase (T
360
).
The cell count was performed by serial dilution and plating
in MRS agar (Difco). The plates were incubated at 37 °C
for 48 h under anaerobic conditions (Anaerobac, Probac,
São Paulo, Brazil). The commercial probiotic L. rhamnosus
GG (ATCC 53103) was used as a reference strain. The
assay was repeated on three independen t occasions in
duplicate.
Bile salt hydrolase activity
Fresh bacterial cultures of the studied lactobacilli (8.0–9.0 log
CFU/mL) were screened for bile salt hydrolase (BSH) activity
as previously described by de Paula et al. (2014) using MRS
plates supplemented with taurodeoxycholic acid sodium salt
(TDCA) or taurocholic acid sodium salt hydrate (TC); MRS
plates without TDCA and TC were used as negative controls,
whereas L. mesenteroides SJRP 55 was used as a positive
control. The plates were incubated anaerobically at 37 °C for
48 h. The presence of precipitated bile acid around the spots
was considered a positive result (Rodríguez et al. 2012). The
assay was repeated on three independent occasions in
triplicate.
Adhesion to Caco-2 cells
The Caco-2 cell line BCRJ 0059 (Rio de Janeiro Cell Bank,
Rio de Janeir o, Brazil) was cult ured (passa ges 29–31) in
Dulbecco’s modified Eagle’s minimum (DMEM, Sigma-
Aldrich) supplemented with 10% heat-inactivated fetal bovine
serum (Cultilab, Campinas, Brazil), a mixture of penicillin
(100 UI/mL) and streptomycin (100 μg/mL) ( Sigma-
Aldrich), and 1% non-essential amino acid solution (Sigma-
Aldrich) at 37 °C in a 5% CO
2
atmosphere.
The adhesion assay was performed as described by Argyri
et al. (2013). All bacterial cultures were grown for 18 h in
MRS at 37 °C before the assays, harvested by centrifugation
(7000 g, 7 min, 5 ° C), washed twice with phosphate-buffered
saline (PBS) and re-suspended in DMEM without any serum
or antibiotics. The commercial probiotic L. rhamnosus GG
(ATCC 53103) was used as a reference strain. Subsequently,
1 mL containing approximately 8.0–9.0 log CFU bacterial
cells was added to each well, and each strain was evaluated
for adherence in duplicate wells in each experiment. After
incubation for 2 h at 37 °C, the cells were washed three times
with sterile PBS to remove non-adherent bacteria, and then
detached from each well by the addition of 1 mLTriton X-100
(0.5% v/v) (Sigma-Aldrich). Following incubation for 5 min
at 37 °C, the cell lysates were serially diluted and plated on
MRS agar. Bacterial adhesion (%) was calculated by the ratio
of adhered bacteria to the total number of added bacteria. The
experiment was performed on three independent occasions.
Presence of genes encoding adhesion, aggregation
and colonization factors
The investigated Lactobacillus strains were tested for the pres-
ence of adhesion , aggr egation and colonizatio n genes
(Table 2 ) a s described in the section BPresence of genes
encoding virulence factors, antibiotic resistance and biogenic
amines^.
Ann Microbiol (2017) 67:289–301 293