LWT - Food Science and Technology 84 (2017) 271e280

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LWT - Food Science and Technology

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Identification, characterization, and probiotic potential of Lactobacillus

rhamnosus isolated from human milk
Muhammad Shahid Riaz Rajoka a, Hafiza Mahreen Mehwish c, Muhammad Siddiq b,
Zhao Haobin a, Jing Zhu a, Li Yan a, Dongyan Shao a, Xiaoguang Xu a, Junling Shi a, *
a
Key Laboratory for Space Bioscience and Space Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, Shaanxi,
People's Republic of China
b
Department of Neonatology, The First Affiliated Hospital, College of Medicine, Xi'an 710072, Shaanxi, People's Republic of China
c
Department of Biotechnology, University of Agriculture Faisalabad, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Breast milk is a source of lactic acid bacteria with remarkable functional and Technological properties; it
Received 12 December 2016 is also a potential source of probiotics. In the present study, seven strains of Lactobacillus rhamnosus were
Received in revised form isolated from breast milk and identified according to their 16S rDNA sequences. Furthermore, their
20 April 2017
probiotic potential was evaluated. The probiotic properties were tested for aspects of antibiotic sus-
Accepted 23 May 2017
ceptibility, antimicrobial activity, lysozyme tolerance, gut condition tolerance (low pH, bile salt tolerance,
Available online 29 May 2017
and 0.4% phenol resistance), hydrophobicity, antioxidant ability, aggregation ability, and adhesion to
Caco-2. Most isolates were resistant to Streptomycin, Ampicillin, Gentamicin, Kanamycin, Penicillin,
Keywords:
Lactobacillus rhamnosus
Cephalotoxin, and Ciprofloxacin. The isolate shows a strong ability to adhere to Caco-2 cells as well as
Probiotic potential DPPH radical scavenging activity in the range of 76%e85%. Isolates SHA113 and SHA117 showed a high
Breast milk survival rate under gastrointestinal tract conditions (>80%), indicating excellent potential for application
Antioxidant as probiotics. The results of these tests indicate that the lactic acid bacteria isolated from human milk
have excellent potential for use as probiotics in various products.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction € & Peta

According to definition (Food & Organization, 2006), probiotics
are living microbes within hosts that exert health benefits when
ingested in sufficient amounts, regulating the immune system,
maintaining healthy gut microbiota, reducing hypertension,
lowering cholesterol levels, and preventing diarrhea (García-Ruiz
et al., 2014; de Vrese & Schrezenmeir, 2008). The genera of Lacto-
bacillus, Bifidobacteria, Lactococcus, Streptococcus, and several
strains of yeast have been used as probiotics (Ohland &
MacNaughton, 2010; Vidhyasagar & Jeevaratnam, 2013).
Numerous species of these genera are “Generally recognized As
Safe” by the FDA (US food and drug administration) or received the
“Qualified Presumption of Safety” status by the ESFA (European
food authority). To be utilized as probiotic, bacteria must have the
ability to survive in the acidic condition of the stomach and within
* Corresponding author.
E-mail address: sjlshi2004@nwpu.edu.cn (J. Shi).
bile acid at the start of the gastrointestinal tract (Erkkila €ja
€,
2000; Rubio, Jofre , Martín, Aymerich, & Garriga, 2014).
To date, numerous probiotics have been isolated from the hu-
man gastrointestinal tract and from dairy products. A recent
investigation reported that the infectious diseases incidence
reduced in breast fed infants compared to infants who received
other milk products. This indicated that specific factors are present
in breast milk that seem to provide protection against infectious
diseases. Breast feeding is an important source of lactic acid bac-
teria for the infant and the lactic acid bacteria that are present in
mother milk can also protect the infant from pathogenic microbes
(Kozak, Charbonneau, Sanozky-Dawes, & Klaenhammer, 2015;
Martín et al., 2003; Rodríguez et al., 2012).
The isolation of probiotic bacteria from breast milk (exerting its
protective effect via production of hydrogen peroxide and organic
acid) is an attractive approach for the selection of probiotic strains,
as their in vivo production might play an important role in the
prevention of infectious disease in the gastrointestinal tract of the
host (Martín et al., 2005). The aggregation and adhesion ability of

http://dx.doi.org/10.1016/j.lwt.2017.05.055
0023-6438/© 2017 Elsevier Ltd. All rights reserved.
272 M.S. Riaz Rajoka et al. / LWT - Food Science and Technology 84 (2017) 271e280
probiotic microbes is also an important feature as they can form
barriers, thus preventing colonization by pathogenic microbes
(Ciandrini et al., 2016; Fontana, Cocconcelli, Vignolo, & Saavedra,
2015). Numerous studies furthermore evaluated the probiotic po-
tential of lactic acid bacteria isolated from adult and infant feces, for
aspects of their resistance to bile salt, low pH, lysozyme, adhesion
to intestinal cells, and immunomodulatory properties
(Davoodabadi, Dallal, Lashani, & Ebrahimi, 2015; Fernandez et al.,
2014; Halimi & Mirsalehian, 2016; Rubio et al., 2014).
Several reports indicated that lactobacilli strains carry antibiotic
resistance genes that can be transferred to other bacteria in the host
gut. Therefore, the evaluation of the antibiotic resistance properties
of probiotics is very important. Furthermore, characteristics related
to safety, survival in host gut, and colonization abilities are
important for evaluating the proposed probiotic bacteria (Lavilla-
Lerma, Pe rez-Pulido, Maqueda, & Valdivia, 2013; Tejero-Sarin ~ ena,
Costabile, Gibson, & Rowland, 2012).
The objective of the present study was to perform a thorough
screening of Lactobacillus rhamnosus strains from human breast
milk and to evaluate their potential use in probiotic products.
2. Material and methods
2.1. Subjects and sample cultivation
Ten (10) breast milk samples of healthy women (women's age
was in the range of 25e34 years and their infant age was in the
range of 8 dayse45 days) were collected from the first affiliated
hospital of the Xi'an Jiaotong University, Xi'an, Shaanxi, China. The
milk samples were transported within sterile containers and pro-
cessed immediately upon receipt.
2.2. Isolation and screening of lactic acid bacteria isolates
The samples were diluted in PBS and spread on de Man-Rogosa-
Sharpe, supplemented with 0.05% L-cysteine (MRSc) agar plates. 1%
CaCO3 was added to MRSc agar medium to indicate acid producing
bacteria (Chen et al., 2016). The plates were incubated aerobically at
37
C for 72 h. Acid producing colonies were identified by a clear
zone around each colony and the ones with different morphologies
were selected for further purification. Single, pure colonies were
isolated and subcultured for further experiments. Each selected
LAB isolate was subjected to tests of morphology, catalase, and
Gram reaction. The isolates of gram positive, catalase negative, and
rods shape were suspected to be lactobacilli and then maintained in
MRSc supplemented with 20% glycerol at 80
C.
2.3. Identification of lactic acid bacteria
Molecular identification was utilized to identify the obtained
strains. The total Genomic DNA of isolates was extracted using the
Genomic DNA purification kit (TransGen Biotech Co., Ltd., Beijing,
China), following the manufacturer's instructions. The primers used
for amplifying the 16S rDNA sequences are forward 500 -AGAGTT
TGATCC TGG CTC AG-300 and reverse 500 -CCGTCA ATT CCT TTGAGT
TT-3” (Beasley & Saris, 2004). The fragments were amplified in a
Techne-TC 512 Thermocycler (England) under the following con-
ditions: 94
C, 30 cycles of 94
C for 45 s, 55
C for 30 s and finally
72
C for 10 min. The amplified fragment was screened on agrose
gel and sequenced by the GenScript Company, Nanjing, China. All
obtained sequences were screened via the BLAST program (https://
blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were deposited
into Gene Bank. Sequence alignment was performed via ClustalW2
(http://www.ebi.ac.uk/Tool/mas/clustalw2/) and a phylogenetic
tree was constructed via neighbor-joining (Saitou & Nei, 1987) and
maximum-composite likelihood methods (Tamura, Nei, & Kumar,
2004) using Mega 6.0 software (http://megasoftware.net/).
2.4. Survival under GIT conditions
2.4.1. Acid tolerance
The ability of isolates to survive at pH 2.0, pH 3.0, and pH 6.2
(control) was evaluated following the method described by (Oh &
Jung, 2015); acid resistance was evaluated via plate count on
MRSc agar.
2.4.2. Bile salt tolerance
The bile salt tolerance of isolated strains that survived for 3 h in
acidic conditions were determined using MRSc broth containing
0.3%, 0.5%, and 1.0% of bile (w/v), following the method described
by (Oh & Jung, 2015). Resistance was evaluated via plate count on
MRSc agar.
2.4.3. Lysozyme resistance
Tolerance to 100 mg/l lysozyme was assessed following the
method reported by (Dias, Vilas-Boas, Hogg, & Couto, 2015).
2.4.4. Bile salt hydrolase activity
The fresh bacterial cultures were streaked in triplicate on MRSc
agar containing 0.37 g/l CaCl2 and 5 g/l sodium salt of taurodeox-
ycholic acid. The plates were incubated aerobically at 37
C for 3
days. The presence of precipitated bile salt around the colonies was
considered to be a positive result.
2.4.5. Resistance to 0.4% phenol
The ability to grow in the presence of phenol was evaluated
using MRSc broth, supplemented with 0.4% phenol. The enumera-
tion of bacteria was performed at 0 h and after 24 h of incubation at
37
C.
2.5. Autoaggregation
The autoaggregation abilities of the selected isolates were
analyzed according to (Collado, Meriluoto, & Salminen, 2008). LAB
cells were washed twice with PBS, resuspended in the same buffer,
and adjusted OD630 nm ¼ 0.25 ± 0.05. Bacterial cell suspensions
were incubated at 37
C for 24 h with sampling at 2 h intervals and
the percentage was expressed as autoaggregation % ¼ 1-(At/A0);
where A0 represents the absorbance at 0 h and At represent
absorbance at 2, 4, 6, 12, and 24 h.
2.6. Coaggregation
In the coaggregation test, bacterial suspensions were prepared
identical to the autoaggregation analysis above. The same volume
of each LAB isolate and pathogen strain were mixed and incubated
at room temperature for 24 h and the coaggregation percentages
were monitored after incubation according to methods described
by (Del Re, Sgorbati, Miglioli, & Palenzona, 2000).
2.7. Cell surface hydrophobicity
A cell surface hydrophobicity assay using xylene was conducted
following the method reported by (Kotzamanidis, Kourelis,
Litopoulou-Tzanetaki, Tzanetakis, & Yiangou, 2010; Mishra & Pra-
sad, 2005) and xylene was chosen as apolar solvent since it reflects
both hydrophobicity and hydrophilicity (Kos et al., 2003).
 M.S. Riaz Rajoka et al. / LWT - Food Science and Technology 84 (2017) 271e280
2.8. Exopolysaccharide production
LAB isolates were plated on a MRSc agar plate with glucose as
the only carbon source and plates were incubated at 37
C for three
days. Triplicate plates containing 25 to 250 colonies were scored for
their mucoid property, following the method reported by (Manini
et al., 2016).
2.9. Biofilm quantification
This assay was performed in a 96 well microtitter plate ac-
cording to the method described by (Musk, Banko, & Hergenrother,
2005).
2.10. Antibiotic resistance test
LAB isolates were screened for resistance against Streptomycin
(10 mg/ml), Ampicillin (10 mg/ml), Erythromycin (15 mg/ml), Tetra-
cycline (30 mg/ml), Gentamicin (10 mg/ml), Kanamycin (30 mg/ml),
Penicillin (10 mg/ml), Cephalotoxin (15 mg/ml), Ciprofloxacin (5 mg/
ml), and Amoxicillin (30 mg/ml), using the disc diffusion method
described by (Angmo, Kumari, & Bhalla, 2016; Jorgensen &
Turnidge, 2015). After incubation for 24 h at 37
C, the diameter
(mm) of the inhibition zone was measured.
2.11. Antimicrobial activity
Agar well diffusion assays were utilized to test the antimicrobial
activity as described by (Fontana et al., 2015). The 18 h old LAB
cultures were tested against the indicator microorganism E. coli
ATCC 25922, Salmonella enterica Serovar Typhimurium CMCC (B)
50115, and Staphylococcus petrasiisubsp. PragensisKY196531, which
is a local isolate from human breast milk.
2.12. DPPH free radical scavenging ability
The DPPH free radical scavenging ability of isolates was deter-
mined for the supernatants from 24 h MRSc medium cultures.
100 ml of supernatant cultures were mixed with 100 ml of ethanolic
DPPH solution in 96 well plates. The plates were incubated in the
dark for 30 min at room temperature. The absorbance was
measured at 517 nm and the scavenging ability was calculated ac-
cording to the following Equation:
Scavenging ability (%) ¼ [1-(A517 of sample/A517 of blank)]  100
2.13. Adhesion to the Caco-2 cell line
Caco-2 cells were grown in cell culture bottles using DMEM
medium supplemented with 2 mM L-glutamine, 10% heat inacti-
vated fetal bovine serum, 100 mg streptomycin/ml, 1% non-essential
amino acid and 100 Ul penicillin/ml. Caco-2 cells were subse-
quently seeded into 24 wells culture plates at a concentration of
2.5  105 cells per well and allowed to differentiate for three days,
while the medium was changed daily. The cells were incubated at
37
C in 5% CO2 atmosphere. Overnight cultures of the isolates were
centrifuged, washed twice via PBS and resuspended in the same
buffer to an appropriate dilution. After that, the bacterial cells were
added to each well and plates were incubated at 37
C for 4 h. After
incubation, cells were washed with PBS and lysed with 0.1% Triton
273
X-100 solution. The cell lysate was serially diluted and spread on
MRSc agar plates. The plates were incubated at 37
C for three days.
The percentage of bacterial adhesion was calculated according to
the following Equation:
Adhesion% ¼ (adhered bacteria/total added bacteria)  100
2.14. Statistical analysis
Values are presented as mean values and standard deviations of
triplicate experiments. Significant ANOVA results were followed up
with Tukey's Multiple Comparison Test in all assays and differences
were considered statically significant if p < 0.05.
3. Results
3.1. Isolation and identification of Lactobacilli
A total of 40 LAB isolates were isolated from human breast milk.
Out of these, seven isolates showed an appearance of lactic acid
bacteria on MRSc medium supplemented with 1% CaCO3 and were
randomly selected for further experiments. All isolates were gram
positive, catalase negative, rod shaped, and showed a clear zone
around the colonies on MRSc medium supplemented with 1%
CaCO3. All isolates were mesophilic and able to grow at 30
C and
37
C in the presence of 2.5%e5% NaCl concentration within me-
dium. A phylogenetic tree was constructed based on their 16S rDNA
sequences utilizing the neighboring method. The results showed
that the seven strains belonged to Lactobacillus rhamnosus (Fig 1).
3.2. Survival under GIT condition
3.2.1. Acid tolerance
Fig. 2A shows survival rates of the isolated strains at pH 2.0 and
pH 3.0 after 3 h of incubation. At pH 3.0, the survival rate was
higher compared to at pH 2.0 for all tested isolates. The survival
rates of SHA111, SHA112, SHA113, SHA114, SHA115, SHA116, and
SHA117 remained above 81% and 90% after exposure to pH 2.0 to pH
3.0 for 3 h. The results indicate that all tested strains showed high
resistance to acidic conditions. More importantly, the isolates
SHA113 and SHA117 showed high resistance to very low pH values.
3.2.2. Bile tolerance
The results revealed that all isolates tolerated various concen-
trations of bile salt (0.3%, 0.5%, and 1%) during 3 h incubation
(Fig. 2B). However, with increasing bile salt concentration, their
growth rate decreased. The isolate SHA116 showed better tolerance
to 0.3%, 0.5%, and 1% bile salt than any other isolates.
3.2.3. Lysozyme tolerance
The overall resistance of the isolates to lysozyme (100 mg/l) was
expressed as the percentage of survival rate, ranging from a mini-
mum of 49.6% to a maximum of 100%. Isolates SHA113, SHA115,
SHA116, and SHA117 showed high resistance (>84%) after 120 min
of incubation, which was considered as a severe treatment
compared to control (Fig. 2C).
3.2.4. Bile salt hydrolase activity
All isolates showed the ability to hydrolyze the sodium salt of
taurodeoxycholate. This became apparent due to the presence of a
274 M.S. Riaz Rajoka et al. / LWT - Food Science and Technology 84 (2017) 271e280

hole around the colonies after growth in MRSc agar plate supple- 3.5. Exopolysaccharide production
mented with 0.37 g/l CaCl2 with 5 g/l sodium salt of taurodeox-
ycholic acid (Table 1). All isolates had the capability to produce exopolysaccharides
when they grew on MRSc agar plates that were supplemented with
3.2.5. 0.4% phenol tolerance glucose (2% v/v) as a carbon source (Table 1).
All tested isolates showed impactful tolerance to 0.4% phenol
after 24 h incubation (Table 1).

3.6. Biofilm quantification

3.3. Autoaggregation and coaggregation
A 96-wells micro titter plate assay was carried out to quantify
All isolates had high autoaggregation and a clear supernatant
biofilm formation. All isolates were found to form biofilms on the
after 24 h incubation (Fig. 3A). The isolate SHA111 showed the
bottom of the well in MRSc broth under aerobic conditions (judging
highest autoaggregation rate (59%), while SHA113 showed the
by their capacity to retain crystal violet). However, biofilm-forming
lowest (51%). In addition, autoaggregation abilities of all isolates
capability differed among strains (Fig. 5B). Isolates SHA114, SHA116,
increased after 24 h of incubation.
and SHA117 showed high biofilm-forming capability (O.D540 > 1.7),
All isolates showed marked coaggregation with E. coli ATCC
followed by SHA111, SHA112, and SHA115 (O.D540 > 1.0).
25922, S. entericserovar Typhimurium CMCC (B) 50115, and
S. petrasiisubsp. PragensisKY196531) (Fig. 3B, C, D). Among the
tested isolates, SHA117 showed the highest coaggregation capa-
bility with E. coli (49%), Salmonella (52%), and Staphylococcus (29%). 3.7. Antibiotics resistance

3.4. Cell surface hydrophobicity
In general, SHA111, SHA113, SHA114, SHA116, and SHA117 had
higher percentages of hydrophobicity (51%, 68%, 50%, 60%, and 69%)
compared to other isolates. However, isolate SHA115 had the lowest
hydrophobicity (33%) and isolate SHA112 had an intermediate level
of hydrophobicity (Fig. 4B).
The minimum inhibitory concentrations of isolates were
measured for ten antibiotics (Table 2). All isolates were resistant to
Streptomycin, Ampicillin, Gentamicin, Kanamycin, Penicillin,
Cephalotoxin, and Ciprofloxacin and susceptible to Amoxicillin
according to the break point introduced by (Panel, 2012). SHA114
and SHA115 were also susceptible to Tetracycline and
Erythromycin.

Fig. 1. Position of the selected seven isolates in the neighbor joining phylogenetic tree.
The phylogenetic tree was constructed on the basis of 16S rDNA sequences. The scale bar 0.01 indicates the nucleotide substitution rate at each site. Bootstrap probabilities were
determined using 1000 replicates and presented as the percentage values. The numbers in parentheses are the accession numbers of selected sequences. The filled circles indicate
the strains are from NCBI and the Empty circles are the out groups used for tree construction.
 M.S. Riaz Rajoka et al. / LWT - Food Science and Technology 84 (2017) 271e280 275

Fig. 2. Resistance of the selected Lactobacillus rhamnosus isolates against acidic conditions (A), bile salt (B), and lysozyme (C).
(A) The treatments were carried out under pH 2.0 or pH 3.0 for 3 h. The results are the value of mean ± SD from three independent runs. The data are significantly different from that
of the controls (pH 6.2) at the level of p < 0.05. (B) The broth is supplemented with 0.3%, 0.5% and 1% bile salt. The control is MRSc broth without bile salt. The results are expressed
as mean ± SD of three independent replicates. All treatments are significantly different from the control at the level of p < 0.05. (C)The MRS broth was supplemented with 100 mg/l
lysozyme. The control is carried out in MRS broth without lysozyme. All treatments are significantly different from the control at the level of p < 0.05.

3.8. Antimicrobial activity results obtained from the agar well diffusion method varied from
6 mm to 14 mm among different strains. Comparatively, the anti-
All isolates expressed a clear inhibition zone (with a diameter microbial activity of all isolates against E. coli and Staphylococcus
above 15 mm) against the three indicator strains. However, the (8 mme13 mm) was higher than those against Salmonella
276 M.S. Riaz Rajoka et al. / LWT - Food Science and Technology 84 (2017) 271e280

Table 1
Tolerance to 0.4% Phenol, BSH activity and exopolysaccharide production of the selected isolates.

Isolates Tolerance to 0.4% phenol (indicated as the log value of CFU/ml)a BSH activityb Exopolysaccharide productionc

0h 24 h △(24 h-0 h)

SHA111 8.78 ± 0:05 8.25 ± 0:02 0.53 þ þ

SHA112 8.65 ± 0:2 8.89 ± 0:1 0.24 þ þ
SHA113 8.20 ± 0:1 7.89 ± 0:1 0.31 þ þ
SHA114 8.86 ± 0:1 9.89 ± 0:05 1.03 þ þ
SHA115 7.95 ± 0:05 8.13 ± 0:05 0.18 þ þ
SHA116 8.19 ± 0:1 8.65 ± 0:05 0.46 þ þ
SHA117 7.85 ± 0:1 8.54 ± 0:05 0.69 þ þ
a
Values are represented as mean± SD (n ¼ 3).
b
þ, positive in BSH activity, identified as the occurrence of precipitated zone (mm) around bacterial colonies on MRSc agar plates supplemented with 0.37 g/l CaCl2 and 5 g/l
sodium salt of taurodeoxycholic acid.a.
c
þ, positive in exopolysaccharide production, identified as the occurrence of sticky bacterial colonies.

typhimurium (Table 3).
3.9. DPPH scavenging ability
Fig. 5A shows that the isolate SHA112 had the highest DPPH
scavenging ability (88%), followed by SHA115 (85%), SHA116 (85%),
SHA114 (85%), SHA113 (84%), SHA111 (76%), and SHA117 (79%).
3.10. Adhesion to Caco-2 cells
No significant differences were found among different isolates
regarding their adhesion abilities (Fig. 4A). Comparatively, the
isolate SHA113 showed the strongest adhesion ability (82%) to
Caco-2 cells. However, it should be mentioned that other isolates
also showed high adhesion abilities to Caco-2 cells in a range of
63%e76%.
4. Discussion
In infants, the establishment of gastrointestinal tract microbiota
is assumed critical for maintaining health and homeostasis of ani-
mals, including humans. Breast milk is the most important factor
for immunological programming, metabolome, and microbiome
(Aaltonen et al., 2011). It is also an important source of gut micro-
flora for new born babies, since it is the only food babies receive
(Arici, Sagdic, & Ozdemir, 2004). Numerous studies indicate that
the decrease of the abundance of lactic acid bacteria in the gut is
one of the major factors related to the reduction of immunological
capability and metabolic disturbance in adults and aging persons
(Tsai, Cheng, & Pan, 2012). Therefore, microorganisms from breast
milk samples are suggested to aid the maintenance of human
health. To verify this hypothesis and to retain lactic acid bacteria
with high potential for human health, we tested isolates from
different breast milk samples and their potential for health-keeping
in aspects of different conditions similar to that in the stomach and
initial intestines.
Many lactic acid bacteria have been developed as probiotic
microbes to improve the health of animals and humans (Rajoka,
Shi, Zhu, Shao, Huang & Yang, 2017). Lactobacillus rhamnosus was
reported to have multiple functions in the maintenance of human
health, such as modulation of immune system and protection
against invading pathogenic microbes (Kaewiad, Kaewnopparat, &
Kaewnopparat, 2015). However, many studies are still being carried
out to isolate new strains with higher potential, since the currently
obtained strains still have weaknesses in practical application,
especially low survival rate and poor proliferation ability through
stomach and guts. The ability to survive under high bile salt con-
centrations and low pH are important features for the successful
passage through the gastrointestinal tract (Mandal, Jariwala, &
Bagchi, 2015; Oh & Jung, 2015). In this study, seven Lactobacilli
isolates were obtained from the breast milk of healthy mothers and
were selected according to their survival capability under artifi-
cially simulated conditions in digestive systems. Among these
seven, four isolates showed more than 80% survival rate at a bile
concentration of 1.0% (w/v), a level much higher than that typically
used in other studies in which Lactobacillus rhamnosus IMC501 and
Lactobacillus paracasei IMC502 were tested at a bile concentration
of 0.3% (m/v) (Verdenelli et al., 2009). In addition, all seven isolates
selected in the study showed more than 80% survival rate at pH 2.0
and more than 90% at pH 3.0 after 3 h exposure, indicating that they
can survive passage through the digestive systems. Their survival
rate was higher than that of previously reported strains such as
Lactobacillus rhamnosus and Lactobacillus casei under such low pH
values (Manini et al., 2016; Tulumoglu et al., 2013).
The response of Lactobacilli to lysozyme was previously found to
be species and strain dependent (Dias et al, 2015). Our results
revealed that the isolate SHA115 was highly resistant to 100 mg/l
lysozyme (resulting in a survival rate above 91%, which is much
higher than previously reported for Lactobacillus rhamnosus strains
PRA211 and PRA323 (Solieri, Bianchi, Lemmetti, & Giudici, 2014)).
Phenol is a common byproduct of the aromatic amino acid meta-
bolism in the intestine. In our study, all isolates tolerated high
phenol concentrations (0.4%), indicating that they can resist the
bacteriostatic effect of phenol in the intestine (Palaniswamy &
Govindaswamy, 2016).
The properties of cell surface hydrophobicity, autoaggregation,
and coaggregation are related to the adhesion properties of lacto-
bacilli strains and are essential for the protection and colonization
of the gastrointestinal tract. A minimum value of 40% hydropho-
bicity is an essential requirement for a probiotic strain (Del Re et al.,
2000). In this study, the isolates SHA113 (68%) and SHA117 (69%)
showed high hydrophobicity to xylene. They also showed high
autoaggregation (SHA113: 52% and SHA117: 57%) and coag-
gregation abilities with E. coli (SHA113: 42% and SHA117: 49%) and
Salmonella (SHA113: 48% and SHA117: 52%) during a 24 h incuba-
tion period, indicating that these two isolates from breast milk are
suitable for animal and human use. This is consistent with studies
reported by others (Dias, Duarte, & Schwan, 2013; Palaniswamy &
Govindaswamy, 2016).
Exopolysaccharide is a multifunctional compound that has
interesting applications in both pharmaceutical and food industries
(Rendueles, Kaplan, & Ghigo, 2013; Russo et al., 2012). All isolates in
the study showed a ropy phenotype that is clearly related to exo-
polysaccharide production on MRSc agar medium. This is in
agreement with results reported by others (Degeest, Janssens, & De
Vuyst, 2001; Minervini et al., 2010).
Bacterial biofilms are an important factor to understand the
mechanisms of bacteria to adapt to environmental stress and
 M.S. Riaz Rajoka et al. / LWT - Food Science and Technology 84 (2017) 271e280 277

Fig. 3. Aggregation ability (A) and the coaggregation ability of the selected Lactobacillus rhamnosus isolates with E. coli ATCC 25922 (B), Salmonella Typhimurium CMCC (C) and
Staphylococcus petrasii subsp. Pragensis KY196531 (D).
All the results were obtained after 24 h and the values are represented as mean ± SD of three independent replicates. (A) The aggregation ability significantly different between
different isolates at the level of p < 0.05 as measured by Tukey's test. (B) No significant difference was found among different isolates on the data of coaggregation ability at the level
of p < 0.05 as measured by Tukey's test.
278 M.S. Riaz Rajoka et al. / LWT - Food Science and Technology 84 (2017) 271e280
Fig. 4. The adhesion ability of the selected isolates to Caco-2 cells (A) and the hy-
drophobicity ability of the selected isolates (B).
(A) The error bars indicate the SD from three replicate. (B) The data are the mean ± SD
of three independent runs of three analyses. The results are significantly different
among different strains at the level of p < 0.05.
colonize different niches. L. rhamnosus has the ability to form bio-
films in vitro (Jones & Versalovic, 2009). We found three isolates
from mother milk (SHA112, SHA116, and SHA117) that had the
ability to form biofilms on the bottom of 96-well microtitter plates.
This is consistent with the reported results of L. rhamnosus 10863
(Rebeca Martín, Sobero n, Camino, & Sua rez, 2008).
Due to safety considerations, the obtained isolates were also
tested on their capabilities for antibiotics resistance. As a result,
none of the tested isolates were resistant to amoxicillin, while all
strains were resistant to streptomycin, ampicillin, gentamicin,
kanamycin, penicillin, cephalotoxin, and ciprofloxacin. These re-
sults are consistent with previous results that were obtained using
L. rhamnosus GG species (Chang, Zhang, Ke, Jian-Ping, & Xiao-Kui,
2009; Maragkoudakis et al., 2006). The production of antimicrobial
compounds is one of the centrally important features for probiotics
to compete with and exclude pathogens, ultimately to survive in
the intestinal tract and to express probiotic effect in their hosts (M
Carmen Collado, Gueimonde, Sanz, & Salminen, 2005). In this
study, all selected isolates had strong antimicrobial activity against
E. coli, which is in agreement with a previous study reported by
Tulumoglu et al. (2013). Some recent investigations revealed that
L. rhamnosus could act as an antioxidant by releasing some peptide
with the ability to quench oxygen radicals (Siragusa et al., 2007). In
this study, the cell free supernatant culture of isolates SHA112
Fig. 5. DPPH Scavenging ability (A) and the biofilm production of the selected isolates
(B).
All data are the mean ± SD values of three independent experiments of three analyses.
The results are significantly different among different isolates at the level of p < 0.05 as
measured by Tukey's test.
(88%), SHA113 (84%), SHA114 (85%), SHA115 (85%), and SHA116
(85%) showed strong antioxidant activity, which was better than
results found by others (Oh & Jung, 2015; Palaniswamy &
Govindaswamy, 2016).
The ability to adhere to Caco-2 cells is also an important crite-
rion for the selection of probiotic lactic acid bacteria. This charac-
teristic provides beneficial effects, such as immune system
modulation and exclusion of pathogenic microbes (Lee, Puong,
Ouwehand, & Salminen, 2003; Schiffrin, Rochat, Aeschlimann, &
Donnet-Hughes, 1995). Various in vitro model systems have been
developed for the preliminary selection of adhered strains
(Vesterlund, Paltta, Karp, & Ouwehand, 2005). In our study, the
adhesion capability to Caco-2 cells varied from 60% to 84% among
different isolates, which was much better than in previously found
isolates (Caggia, De Angelis, Pitino, Pino, & Randazzo, 2015; M
Carmen; Collado, Jalonen, Meriluoto, & Salminen, 2006).
5. Conclusion
Breast milk is an excellent source of lactic acid bacteria. In this
study, seven L. rhamnosus strains with probiotic potential were
isolated from breast milk of healthy women. All L. rhamnosus
 M.S. Riaz Rajoka et al. / LWT - Food Science and Technology 84 (2017) 271e280 279

Table 2
Antibiotic resistance of the selected isolates.a

Antibiotics SHA111 SHA112 SHA113 SHA114 SHA115 SHA116 SHA117

Streptomycin (10 mg/ml) þ þ þ þ þ þ þ

Ampicillin (10 mg/ml)
Erythromycin (15 mg/ml)
Tetracycline (30 mg/ml)
Gentamicin (10 mg/ml)
Kanamycin (30 mg/ml)
Penicillin (10 mg/ml)
Cephalotoxin (15 mg/ml)
Ciprofloxacin (5 mg/ml)
Amoxicillin (30 mg/ml)
a
Indicated as ‘þ’, resistance and ‘-‘, susceptible.
þ þ
e þ
þ þ
þ þ
þ þ
þ þ
þ þ
þ þ
e e
þ þ þ þ þ
þ e e þ þ
e e e e þ
þ þ þ þ þ
þ þ þ þ þ
þ þ þ þ þ
þ þ þ þ þ
þ þ þ þ þ
e e e e e

Table 3
Antimicrobial activity of the selected isolates against different bacteria (mm).a

Isolates E.coli ATCC 25922 Salmonella enteric Serovar Typhimurium CMCC (B) 50115 Staphylococcus petrasii subsp. PragensisKY196531

SHA111 11 ± 0.5 8 ± 0.1 11 ± 0.3

SHA112 12 ± 0.5 7 ± 0.1 11 ± 0.2
SHA113 10 ± 0.5 6 ± 0.5 9 ± 0.5
SHA114 9 ± 0.4 7 ± 0.6 10 ± 0.5
SHA115 10 ± 0.6 7 ± 0.3 10 ± 0.4
SHA116 8 ± 0.3 9 ± 0:4 11 ± 0.2
SHA117 9 ± 0.5 9 ± 0.5 13 ± 0.2
a
The antimicrobial activity is indicated as the diameter of the inhibiting zone around the isolate colonies. The values are the expressed as mean ± SD (n ¼ 3). The data less
than 5 mm indicate weak antimicrobial activity; 6 mme8 mm indicate strong antimicrobial activity, 9 mme14 mm indicate strong antimicrobial activity.

isolates had a high potential to adhere and pass through the
gastrointestinal tract. Overall, the isolates SHA113 and SHA117 had
comparatively higher potential for practical application as pro-
biotics for humans due to their high capability of antimicrobial
activity, resistance of lysozyme, phenol and antibiotics, production
of expolysaccharides, formation of biofilms, and antioxidant ac-
tivity. Overall, our study indicated that breast milk is an excellent
resource to isolate lactic acid bacteria with outstanding character-
istics as probiotics. However, more in vitro and in vivo investigations
are still required to confirm the beneficial roles of the isolates ob-
tained in this study to human health.
Acknowledgements
This study was supported by the National Key Technology R&D
Program (grant number 2015BAD16B02) and the National Natural
Science Foundation of China (NSFC) (grant Number 31201408 and
grant number 31471718).
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