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Investigation of Lactobacillus spp. bacteria in infants
consuming breast milk and formula and determination of
some probiotic characters
Tuğçe Muslu Çağal1, Esin Kıray2, Ergin Kariptaş3
1Department of Molecular Medicine, Ahi Evran University, Health Sciences Institute, Kırşehir, Turkey; 2Department of Medical Services
and Techniques, Medical Laboratory Techniques Programme, Ahi Evran University, Vocational School of Health Services, Kırşehir, Turkey;
3Department of Medical Microbiology, Samsun University, Faculty of Medicine, Samsun, Turkey
ABSTRACT
Objectives: It was aimed to investigate some probiotic properties of Lactic Acid Bacteria (LAB) isolated from
stool samples taken from 45 healthy 0-4 month old babies, who did not take antibiotics or probiotic supplements
in the last 3 months, and who did not have any health problems.
Methods: Six different species were obtained from 21 isolates selected by the method of Mass Spectrometry
(MALDI-TOF MS).
Results: The most common strain was Lactobacillus rhamnosus with a rate of 59%, followed by Lactobacillus
paracasei with a rate of 13.6%. Vancomycin, tetracycline, gentamicin, netilmicin, tobramycin, penicillin,
ampicillin, teicoplanin and amikacin antibiotics were used to evaluate the antimicrobial activities of the strains.
In our study, while all strains were resistant to the antibiotic amikacin, they were sensitive to tetracycline,
penicillin, gentamicin, netilmicin, teicoplanin, vancomycin, ampicillin and tobramycin antibiotics. In the
evaluation of the antagonistic activities of LAB, 6 different pathogens (Escherichia coli ATCC 25922,
Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, S. aureus ATCC 25923, Candida albicans
ATCC 10231, Listeria monocytogenes ATCC 19111) were used and it was determined that the strains showed
antimicrobial effects on all pathogenic microorganisms. Cholesterol assimilation abilities, T21 and T22 strains
achieved the highest cholesterol assimilation rate of 39.1%.
Conclusions: It is thought that most of the isolated strains have probiotic potential, and especially Lactobacillus
gasseri T21 and Lactobacillus paracasei T22 strains may be probiotic strains that can be used in the production
of preparations alone or together with other Lactobacillus strains.
Keywords: Lactic acid bacteria, breast milk, probiotic
e-ISSN: 2149-3189
The European Research Journal 2023
DOI: 10.18621/eurj.1202118
Original Article
Molecular Medicine
Address for correspondence: Tuğçe Muslu Çağal, MD., Assistant Professor, Ahi Evran University, Health Sciences Institute, Department of Molecular
Medicine, Kırşehir, Turkey. E-mail: dyttugcemuslu_1993@hotmail.com, Phone: +90 358 211 16 23
©Copyright © 2023 by Prusa Medical Publishing
Available at http://dergipark.org.tr/eurj
info@prusamp.com
Received: November 10, 2022; Accepted: February 13, 2023; Published Online: April 6, 2023
How to cite this article: Muslu Çağal T, Kıray E, Kariptaş E. Investigation of Lactobacillus spp. bacteria in infants consuming breast milk and formula
and determination of some probiotic characters. Eur Res J 2023. DOI: 10.18621/eurj.1202118
One of the most basic events that shape the intes-
tinal microbiota is breastfeeding [1]. Breast milk;
Milk containing 10-105 CFU of microorganisms per
ml from which Streptococcus and Staphylococcus are
the most, Lactobacillus, Bifidobacterium, Enterococ-
cus and short-chain fatty acid producing bacteria such
as Veilonello, Propionibacterium, Faecalibacterium
are easily isolated. Human milk plays an important
Early Online
The European Research Journal 2023 1
Eur Res J 2023 Microbiota in breastfed and formula-fed infants
role in the development of the neonatal gut microbiota,
as it provides a continuous supply of microorganisms
to the infant gut a few weeks after birth [2].
An effective probiotic agent is known to be mi-
croorganisms that can survive the host's digestive
process, colonize the gut, and produce a beneficial re-
sponse in the host without pathogenic or toxic side ef-
fects [3]. Each probiotic strain is known to have its
own characteristics and should be investigated in de-
tail in this context. It is necessary to determine the
types of probiotic characters to be used especially in
infant formula.
For this purpose, in our study, it was aimed to de-
termine the various probiotic characterizations of lac-
tic acid bacteria (LAB) isolated from the stools of 0-4
month-old infants who received only breast milk and
supplemented with formula in addition to breast milk.
METHODS
Isolation of Bacteria
In our study, isolated from the stools of 51 healthy ba-
bies who were admitted to Sivas Cumhuriyet Univer-
sity Health Services Application and Research
Hospital, aged 0-4 months, born vaginally, who did
not receive antibiotic and/or probiotic treatment in the
last 3 months, took only breast milk or were fed with
formula in addition to breast milk. The obtained LAB
was used. During the collection of stool samples, the
baby's gender, week of birth, birth weight, current
weight, feeding style (whether he took only breast
milk or infant formula in addition to breast milk) and
the number of daily defecations were obtained. The
ethics committee of our study was received from
Kırşehir Ahi Evran University on 24.09.2019 (Deci-
sion No 2019-16/165). The samples were brought to
Sivas Cumhuriyet University Faculty of Medicine,
Department of Medical Microbiology, Microbiology
Laboratory, under sterile conditions, and evaluated
within the same day. MRS (De Man Ragosa Sharpe,
Merck) broth and solid media were used in the devel-
opment and activation of LAB. Each sample was in-
cubated in MRS solid medium for 48 hours at 37°C in
anaerobic conditions. Large, small, white, off-white
and opaque colonies formed on the media as a result
of incubation were selected and stored in TSB (Tryptic
Soy Broth) medium with glycerol at -18°C until stud-
ied. Escherichia coli ATCC 25922, Staphylococcus
aureus ATCC 6538, Bacillus subtilis ATCC 6633, S.
aureus ATCC 25923, Listeria moncytogenes strains
used to determine the antagonistic effects of LAB
were obtained from Amasya University Microbiology
Laboratory Culture Collection. Species of isolated
strains were determined by Matrix Assisted Laser Des-
orption Ionization Time of Flight Mass Spectrometry
(MALDI-TOF MS) (Bruker Biotyper 3.0 Mikroflex
LT Bruker Daltonics GmbH Bremen Germany).
MALDI-TOF MS detects the protein profiles of exist-
ing microorganisms and compares the obtained pro-
files with the microorganisms in the current library [4].
Determination of Probiotic Properties of LAB
Acid and Bile Tolerance
In order to determine the accessibility of LAB iso-
lated from the stool of infants to the intestines by sur-
viving in the acidic environment of the stomach, an
environment similar to the gastric fluid environment
was prepared. Cultures activated in broth for 18 hours
were precipitated by centrifugation at 3000 × g for 15
minutes at 4⁰C. After the precipitate was washed twice
with sterile phosphate buffered saline (PBS), PBS
buffer (1N NaOH and 1N HCl) was prepared in 3 dif-
ferent ways with pH levels of 2.0, 2.5,3.0 and the
strains were incubated in low pH PBS buffers for 3
hours at 37⁰C. has been done. PBS with a pH of 7.2
was used for the control. Serial dilutions were made
by taking 1 ml from the samples that were incubated
at low pH at the 0th and 3rd hours of the incubation,
and they were incubated by making triple parallel cul-
tivations on MRS agar media. At the end of the period,
the colonies in the control and test groups were
counted and the % viability rates were calculated. %
Viability = X/X0 (X: Number of viable microorgan-
isms in the test group, XO: Number of viable microor-
ganisms in the control group) [5].
Bile Salt Tolerance: 0.3% by weight to determine the
viability of the isolated LAB in a medium containing
bile salt; MRS broths containing 0.5% and 1% bile
salts (oxgall, sigma) were prepared and the other
stages of the study were applied as stated above.
% Vitality = X/X0 ×100 [6].
pH Change
Each sample was inoculated into MRS broth, and
after 18 hours of incubation at 37⁰C, it was measured
2 The European Research Journal 2023
Eur Res J 2023 Muslu Çağal et al
with a pH meter (AZ Instrument) to determine the
acidic pH values of the cultures. As a control, the pH
of sterile MRS broth without inoculation was checked.
Measurements were carried out in 3 repetitions [7].
Determination of Antibiotic Sensitivities
Activated cultures in MRS medium were adjusted
to 0.5 McF (625 nm absorbance = 0.08-0.1) with phys-
iological saline and spread homogeneously on sterile
MRS agar with a sterile drigalski spatula. Antibiogram
discs were placed in Petri dishes at appropriate inter-
vals and incubated at 37°C for 24 hours. The diameters
of the zones formed around the antibiotic discs as a re-
sult of incubation were measured in millimeters with
a caliper. Measurements were evaluated as Resistant
(R), Semi-Fine (I), and Sensitive (S) according to
NCCLS (National Committee for Clinical Laboratory
Standards) criteria. Ampicillin (AM) (10 mcg) in the
study; Penicillin (P) (10 U); Teicoplanin (TEC) (30
mcg); Gentamicin (CN) (120 mcg); Tetracycline (T)
(30 mcg); Netilmicin (NET) (30 mcg); Vancomycin
(VA) (30 mcg); Tobramycin (TOB) (10 mcg);
Amikacin (AK) (30 mcg) discs were used.
Determination of Lactic Acid Amounts
5 mL of distilled water and 250 microliters of phe-
nolphthalein were added to each of the 5 mL samples
activated for 18 hours at 37 C in MRS broth. It was
titrated by adding 0.1 N NaOH dropwise. The number
of drops is multiplied by 4. The lactic acid content of
each sample was determined so that each 1 ml of 0.1
N NaOH consumed was equivalent to 0.009 g of lactic
acid [8, 9].
Determination of Cholesterol Assimilation Capaci-
ties
In order to determine the cholesterol assimilation
capacities of LAB isolated from the stool microflora
of healthy infants, the total cholesterol level of serum
collected from patients with serum cholesterol level
of 250-300 mg/dL who applied to Amasya University
Sabuncuoğlu Şerefeddin Training and Research Hos-
pital was measured. 1 mL of cultures activated for 18
hours in MRS broth was taken and added to [0.3%
(Oxgall, Sigma)] MRS broth (3 mL) containing bile
salt. Each sample, 1 mL of sterile serum, was collected
into 5 ml tubes by passing it through a 0.45 micro m
disposable (Milipore, USA) filter with a final concen-
tration of 100 mg/mL. After incubation at 37oC for 24
hours, the final cholesterol values of the supernatants
were determined by centrifugation at 5000 × g at 4oC
for 10 minutes. Measurements were carried out in the
Biochemistry Laboratory of Amasya University
Sabuncuoğlu Şerefeddin Training and Research Hos-
pital on a Roche HITACHI cobas 8000 device. Cho-
lesterol reduction rates were determined by comparing
the cholesterol amount of the samples before incuba-
tion with the amount of cholesterol after incubation.
RESULTS
Collection of Stool Samples and Identification of LAB
Only commercial infant formula, who applied to Sivas
Cumhuriyet University Hospital Pediatrics outpatient
clinic for routine controls, was born vaginally, did not
use antibiotics and probiotics in the last 3 months, did
The European Research Journal 2023 3
!
!
!
Fig 1. Gram stain images of 3 different species isolated from stool microflora, respectively (A) L. rhamnosus, (B) P. acidilactici,
and (C) L. reuteri.
Eur Res J 2023 Microbiota in breastfed and formula-fed infants
not have a history of hospitalization, took only breast
milk, received and/or receives commercial infant for-
mula in addition to breast milk. For LAB isolation, 22
gram positive, white, opaque-looking pure isolates
were selected. It was observed that the babies from
whom 22 selected isolates were obtained were 36 days
old on average.
Gram stain images of some isolates are given in
Fig. 1. Identification of isolates was performed using
MALDI-TOF MS.
According to the MALDI-TOF MS method,
which is based on looking at the protein profiles of mi-
croorganisms (protein, peptide, sugar) and large or-
ganic molecules after they are ionized and passed
through electric and/or magnetic fields, Lactobacillus
rhamnosus is the most common strain with a rate of
59%. Lactobacillus paracasei follows with 13.6%.
Then there were 9% Lactobacillus reuteri, Lactobacil-
lus gasseri and 4.5% Enterococcus faecalis, Pediococ-
cus acidilactici strains. Gram stain images of some
species are given in Fig. 1.
Determination of Probiotic Properties of LAB
Acid and Bile Tolerance of Stool LAB
Determination of resistance to acid and bile salts
of LAB isolated from stool swabs was performed in
vitro by exposing the strains to pH (pH 2.0, 2.5 and
3.0) adjusted PBS. The survival rates of LAB in pH
4 The European Research Journal 2023
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Eur Res J 2023 Muslu Çağal et al
medium are given in Table 1 and the survival rates in
MRS medium containing low bile salt are given in
Table 2.
pH Change
After 18 hours of incubation at 37oC in MRS
broth, the mean pH values of L. rhamnosus strains
were 4.36; The average pH value of L. reuteri strains
was 4.04; The mean pH value of L. paracasei strains
was 4.03; The average pH value of L. gasseri strains
was measured as 3.9.
Determination of Antibiotic Sensitivities
In our study, while T3 and T18 strains were sus-
ceptible and semi-susceptible to ampicillin antibiotic,
all other strains were resistant; T2, T12, T13, T17, T18
strains were resistant to Penicillin antibiotic, while all
other strains were found to be susceptible. Although
resistance to antibiotics varies according to strains, re-
sistance to ampicillin was 91% for all strains; resist-
ance to teicoplanin 59%; gentamicin resistance 36%;
resistance to netilmicin 54%; vancomycin resistance
64%; tobramycine resistance 95%; resistance to
amikacin was observed at a rate of 100%
Determination of LAB's Lactic Acid Amounts and
Cholesterol Removal
The amount of rubbery acid was calculated with
The European Research Journal 2023 5
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Eur Res J 2023 Microbiota in breastfed and formula-fed infants
the help of phenolphthalein and NaOH for the cultures
activated in MRS broth at 37⁰C for 18 hours. The av-
erage amount of lactic acid produced by LAB and pro-
biotic bacteria after being activated in MRS broth at
37⁰C for 18 hours is 361.2. It was observed that L.
rhamnosus T11 strain had the highest lactic acid pro-
duction capacity with 496.8. L. paracasei T17 strain
had the lowest lactic acid production with 86.4.
Among the strains used in our study, the strains
with the highest cholesterol assimilation capacity were
T21 and T22 strains with a rate of 39.1%. These
strains are followed by T18, T16, T17, T17, T4, T9,
T13 strains. The strain with the least cholesterol as-
similation capacity was found to be the T20 strain. The
lactic acid amounts and cholesterol removals of LAB
are shown in Table 3.
DISCUSSION
There are many factors that shape the baby's mi-
croflora immediately after birth. Some of those; The
birth type of the baby, health and immunological sta-
tus, whether the baby receives breast milk, the
mother's diet, the GIS (Gastro-intestinal System) tran-
sition time and GIS pH are factors such as stress [10].
In another study evaluating the microbiota of in-
fants fed with formula (only or with breast milk), the
bacterial groups that colonize formula-fed infants
more frequently were Enterococcus, C. coccoides,
Atopobium cluster, B. vulgatus, B. longum subsp.
longum is indicated [11].
E.coli, C. difficile, B. fragilis species are less in
breastfed infants, but Bifidobacteria, especially B.
6 The European Research Journal 2023
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Eur Res J 2023 Muslu Çağal et al
breve and B. longum, are early colonizers in infants,
but B. animalis subsp. lactis occurs only with the type
of diet and is not the common infant gut microorgan-
ism [12, 13].
Similar to our study, fecal Bifidobacterium and
Lactobacillus/Enterococcus counts were found to be
higher in breastfed infants compared to formula-fed
infants at 6 months [14]. L. acidophilus (20%), L. aci-
dophilus-3 (10%), L. brevis (30%), L. casei (15%)
bacteria were isolated from colostrum. L. brevis
(41.2%), L. fermentum (11.8%), L. reuteri (5.9%), L.
rhamnosus (11.8%), L. plantarum (29.4%) were de-
tected in stool [15]. The results of the study are similar
to the results of our study. In our study, stool samples
were collected from 45 healthy babies and 22 bacterial
isolates were evaluated. 54.5% of the babies to whom
these bacterial isolates belong are exclusively breast-
fed; While 31.8% are fed with commercial infant for-
mula, 13.6% are fed with infant formula as well as
breast milk. Of the strains isolated from the stools of
exclusively breastfed babies, 41.6% were identified as
L. rhamnosus, 16.6% L. reuteri, 25% L. paracasei,
12.5% L. gasseri, 12.5% P. acidilactici. Of the strains
isolated from the stools of infants fed only infant for-
mula, 62.5% L. rhamnosus, 12.5% L. paracasei,
12.5% L. gasseri, 12.5% E. faecalis; All of the strains
isolated from the stools of infants who received infant
formula in addition to breast milk were identified as
L. rhamnosus. Similar to the studies, lactic acid bac-
teria were also found in our study. However, the stools
of exclusively breastfed babies have greater microbi-
ological diversity.
In a study of L. rhamnosus, L.casei, L. paracasei
strains obtained from different sources (meat and dairy
products, sourdough doughs, wine, beverages, vegeta-
bles and human body), survival and growth were im-
proved after exposure to low pH values for 2 hours.
61% of strains evaluated for their restart capacity 2
hours at pH =2.5; 3.3% continued to grow after incu-
bation at pH = 1.5. All strains survived in the presence
of 1.5% bile salt after 24 hours of incubation at 37 °C
[16].
The viability rates of L. rhamnosus 19 strain obtained
from Kenya's traditional fermented food (two) at pH
=2.0 decreased from 7.29 log10 cfu/ml to 4.11 log10
cfu/ml at the end of the 3rd hour. L. rhamnosus 19
strain was incubated at pH = 2.0 for 3 hours, and the
survival rate of L. rhamnosus in MRS broth with
added 0.3% bile salt was reported to be 3.85 log10
cfu/mL at the end of 48 hours [17].
In one study, a total of 22 Lactobacillus strains
isolated from infant feces, low pH and resistance to
bile salts, as well as 8 isolates (L. reuteri 3M02, 3M03;
L. gasseri 4M13, 4R22, 5R01, 5R02, 5R13; L. rham-
nosus 4B15) were evaluated.) has high tolerance to
acids (99.1%) and bile salts (99.9%) [18]. It was stated
that there was no significant decrease in the amount
of P. acidilactici strain kept in acidic salt solutions (pH
= 2.0) [19].
107 lactic acid bacterial isolates were isolated
from 6 donor infant meconium from the Roubaix hos-
pital in northern France. Some of the E. faecalis strains
produced lactic acid up to 7.06g/l after 24 hours of in-
cubation [20]. In our study, the lactic acid amount of
E. faecalis strain obtained from a baby who received
formula was 453.6 mg/dL, and lactic acid production
was below this result. In another study, the average
amount of lactic acid produced by L. rhamnosus
strains obtained from vaginal swabs was 585 mg/dL;
The average amount of lactic acid produced by L.
paracasei strains was 458 mg/dL; The average lactic
acid content of P. acidilactici strains was found to be
682 mg/dL [21]. In our study, while the lactic acid pro-
duction amount of L. rhamnosus strains was found to
be 360 mg/dL on average, it was determined that the
strain with the highest lactic acid production capacity
was L. rhamnosus T11 with 496.8.
In our study, it was determined that LAB isolated
from baby feces was resistant to low pH environment
and showed 100% viability especially in pH 2.5-3.0
environments. It was observed that especially L. rham-
nosus strains survived better in pH 2.0 environment.
L. paracasei strains isolated from Italian Castelmagno
cheese after 24 hours of incubation, mean pH values
of L. paracasei strains were found to be 3.87.30 Av-
erage pH values of L. rhamnosus strains isolated in
our study were 4.36; The average pH value of L.
reuteri strains was 4.04; The mean pH value of L.
paracasei strains was 4.03; The average pH value of
L. gasseri strains was measured as 3.9. [22].
L. rhamnosus, L. paracasei strains obtained from
human faeces showed resistance to vancomicin, col-
istin sulfate, oxalinic acid, gentamicin, oxalinic acid,
kanamicin under high in vitro conditions [23]. In our
study, L. rhamnosus, L. rhamnosus, L. paracasei
strains were similarly resistant to vancomicin but
The European Research Journal 2023 7
Eur Res J 2023 Microbiota in breastfed and formula-fed infants
highly sensitive to gentamicin. In our study, while P.
acidilactici was resistant to ampicillin, tobramycin,
amikacin, it showed sensitivity to penicillin and te-
icoplanin. These results are similar to other studies
[24, 25]. In our study, the E. faecalis strain isolated
from a baby who received only formula was found to
be resistant to all antibiotics used in the study. Simi-
larly, in other studies, it was determined that the E. fae-
calis strain was resistant to many antibiotics [26, 27].
L. paracasei subsp. paracasei (41), L. fermentum
(24), L. rhamnosus (11), L. casei (17), Lactobacillus
spp. (11) strains have been reported to have strong an-
timicrobial effects, especially on S. aureus. 28 L.
monocytogenes ATCC 3512, L. innocus 103982, B.
subtilis ATCC 6633, S. aureus ATCC 3386, E. coli
CIPI 103982 have been reported to show antagonistic
activity against pathogenic bacteria [18].
It is quite remarkable that LAB has potential cho-
lesterol-lowering effects in recent studies [28]. High
cholesterol is associated with cardiovascular diseases,
a major cause of death worldwide. Current therapeutic
measures, lifestyle changes, dietary interventions,
pharmaceutical agents are insufficient to regulate cho-
lesterol level. Probiotic bacteria show the potential to
lower cholesterol levels through inhibition of 3-hy-
droxy-3-methylglutaryl coenzyme A enzymes by dif-
ferent mechanisms, including bile salt hydrolase.
In the study, a total of 22 Lactobacillus strains iso-
lated from infant feces were evaluated, 8 isolates (L.
reuteri 3M02, 3M03; L. gasseri 4M13, 4R22, 5R01,
5R02, 5R13; L. rhamnosus 4B15), especially L. rham-
nosus 4B15 and L. gasseri 4M13 strains have been
shown to have significant cholesterol-lowering capac-
ity compared to other strains [18].
In one study, it was stated that L. rhamnosus strain
showed less cholesterol assimilation with a cholesterol
assimilation value of 13.21% compared to L. aci-
dophilus, L. fermentum, B. Lactis [29]. In our study,
L. rhamnosus, L. reuteri, L. gaseri, L. paracasei.
Among the strains, the strains with the highest choles-
terol removal were L. gasseri (39.1%) and L. paraca-
sei (39.1%) isolated from exclusively breastfed
infants. The mean cholesterol assimilation percentage
of the L. paracasei strain was 28.5%; The mean cho-
lesterol assimilation percentage of the L. reuteri strain
was calculated as 31.05%. With these values, previous
studies showed, on average, higher cholesterol re-
moval.
Limitations
The biggest limitation of our study is that the study
is limited to Sivas province and it is necessary to study
with more stool samples in order to fully reveal the
microbial difference between breast milk and formula
babies.
CONCLUSION
In the light of our results, it is thought that the isolated
strains have strong probiotic potential, especially L.
gasseri T21 and L. paracasei T22 strains, which can
be used in the production of preparations alone or to-
gether with other Lactobacillus strains. In recent years,
with the addition of prebiotics and probiotics to com-
mercial infant formulas, the difference between infants
receiving breast milk and infant formula has de-
creased. However, breast milk, which is a cheap, reli-
able, natural food that meets all the needs of the baby
on its own, should always be the first choice. It is im-
portant to evaluate the use of strains as oral prepara-
tions clinically well.
Authors’ Contribution
Study Conception: TMC, EK, EK; Study Design:
TMC, EK, EK; Supervision: TMC, EK, EK; Funding
TMC, EK, EK; Materials: TMC, EK, EK; Data Col-
lection and/or Processing: TMC, EK, EK; Statistical
Analysis and/or Data Interpretation: TMC, EK, EK;
Literature Review: TMC, EK, EK; Manuscript Prepa-
ration: TMC, EK, EK and Critical Review: TMC, EK,
EK.
Conflict of interest
The authors disclosed no conflict of interest during
the preparation or publication of this manuscript.
Financing
The authors disclosed that they did not receive any
grant during conduction or writing of this study.
Acknowledgements
This article describes Lactobacillus spp. in breast-
fed and non-breastfed infants. It was prepared from
the master's thesis named research of bacteria and de-
termination of some probiotic characters.
8 The European Research Journal 2023
Eur Res J 2023 Muslu Çağal et al
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