BIODIVERSITAS ISSN: 1412-033X

Volume 18, Number 3, July 2017 E-ISSN: 2085-4722

Pages: 1265-1269 DOI: 10.13057/biodiv/d180352

Characterization of Bifidobacteria from infant feces with different

mode of birth at Purwokerto, Indonesia

PANCRASIA MARIA HENDRATI♥, DYAH FITRI KUSHARYATI, DINI RYANDINI, OEDJIJONO

Faculty of Biology, Universitas Jenderal Soedirman. Jl. Dr. Soeparno 63, Purwokerto, Banyumas 53122, Central Java, Indonesia.
Tel.: +62-281-638794, Fax.: +62-281-631700, ♥email: [email protected]

Manuscript received: 30 April 2017. Revision accepted: 30 July 2017.

Abstract. Hendrati PM, Kusharyati DF, Ryandini D, Oedjijono. 2017. Characterization of Bifidobacteria from infant feces with
different mode of birth at Purwokerto, Indonesia. Biodiversitas 18: 1265-1269. Bifidobacteria belongs to the so-called beneficial
intestinal flora. Before attempting to raise their intestinal levels to improve the health status of the host, it is importance to know about
physiological variations in the Bifidobacterial colonization of the human intestine. Birth process influenced the diversity of
Bifidobacteria in infant feces. This research was intended to isolate and characterize Bifidobacterium spp. as well to evaluate their
presence in the feces of infants who were born by mode of normal, caesarean and premature. The research was conducted by survey
method and data were analyzed with descriptive analysis. Bifidobacterium character was observed include colony and cell morphology.
The biochemical test included catalase, oxidase, indole, Voges-Proskauer, different pH growth, and resistance to lysozyme.
Bifidobacterium metabolites obtained tested its bacterial activity to Salmonella typhimurium and Escherichia coli. The result of this
research showed that 35 isolates are suspected Bifidobacterium group and after API 20 A test showed 17 isolates are rally genera of
Bifidobacterium spp.and all isolates come from infant feces with caesar and premature delivery. These isolates inhibited the growth of S.
typhimurium and E. coli with different inhibitory capabilities. This finding is very important for science and medical point of view and
could be developed with further research.

Keywords: Bifidobacteria, Characters, Mode of birth, Infant feces

INTRODUCTION by birth process (Mariat et al. 2009). During vaginal birth

process (normal), the contact with vaginal and intestinal
Probiotics are living microorganisms that provide flora are important resources to begin the colonization in
health benefits to the host when ingested in adequate infants, while during a cesarean birth process, direct
amounts (Bermudez-Brito et al. 2012). In recent years, contact of infant’s mouth with vaginal and intestinal
many studies have shown that probiotics can cure a variety microbiota are not happen, and the intestinal colonization is
of diseases, such as diarrhea, colitis, urinary tract infection, derived from the skin of his parents, workers, and medical
hypertension, allergies, and cancer. Probiotics benefits are equipment (Lif Holgerson et al. 2011).
important from an early age. The previous few years, the The composition of the microbiota at the beginning of
safe test of probiotic bacteria are on the group of human life can affect the health for several months.
Bifidobacteria, i.e. Bifidobacterium lactis. The probiotics Biasucci et al. (2008) stated that the composition of
use for children was focused on the prevention of diarrhea microbiota in infants plays an important role in the
and allergies as well as increasing the number of development of the post-partum immune system. The
Bifidobacteria in the gut. Bifidobacteria is the dominance development of fecal microbiota in neonates is important
microorganism in the human digestive tract of adults and because those bacteria are the first to colonize the sterile
infants (Holzapfel et al. 2001). The dominance of intestine of the neonates and, thus, have a significant effect
Bifidobacteria in the human digestive tract is about 10% in on the host. Bifidobacteria are predominant in the fecal
adults and up to 90% in infants (Harmsen et al., 2000a) that microbiota of infants, and, therefore, they are important to
were analyzed from stool samples. Bifidobacteria colonize an understanding of how commensal Bifidobacteria is
the intestine of infants during birth and the first week after established in the intestine of infants (Mikami, Kimura, and
birth (Heidarpour et al. 2008). Narayanan and Subramonian Takahashi.2012). The important of Bifidobacterium is the
(2015) showed that there are about 29 Bifidobacteria ability to produce antimicrobicrobial compounds called
species that have been identified, including eleven species bacteriocin (Aly et al. 2006).
isolated from infant feces. The most frequently isolated The problem of this research is “how is the dominance
Bifidobacteria species in infant feces are B.bifidum, of Bifidobacterium sp. from the feces of infants with a
B.longum, B. infantis and B. breve. These organisms are different birth process (normal, cesarean, and premature)?”.
Gram-positive, non-motile, non-spore forming, anaerobic, Information of Bifidobacterium isolated from newborn
and pleomorphic rods. Some researchers suspect that the feces is still lack in Indonesia. This reason encourages
presence of intestinal Bifidobacteria in infants is affected researcher to explore Bifidobacterium from infant feces
1266 B I O D I V E R S I T A S 18 (3): 1265-1269, July 2017

especially in Purwokerto. The research on Bifidobacteria as Sulphide Indole Motility Agar (SIMA) medium, and then
the agent of probiotics is currently being developed by was incubated at room temperature for 2x24 hours. The
isolating the potential isolate from infants feces with the spread of growth was observed. A positive result was
different birth process. Vaginal birth process and interpreted with pellicle formation on the surface of the
breastfeeding will stimulate the presence of Bifidobacteria medium.
in the gastrointestinal tract of infants. The research was
intended to isolate and characterize the Bifidobacterium Biochemical test
spp. as well as to evaluate their presence in the feces of infants Catalase test. One loop of the single colony was
who were born by modes of normal, caesarean and premature. smeared on glass object, and then was dropped with the
H2O2 reagent. A positive result was interpreted with gas
bubbles formation.
MATERIALS AND METHODS Test indole. One loop of single colony was inoculated
into indole medium (Tryptone Broth) for 2x24 hours at 35-
The research was conducted at Laboratory of 360C. Then, the medium was added with Kovac’s Indole
Microbiology, Faculty of Biology, Jenderal Soedirman reagent. Positive the result was interpreted with pink rings
University using survey method. The feces were taken formation on the surface of the medium.
from 10 until 12 days old infant with the different birth Voges-Proskauer test. One loop of the single colony
process (normal, cesarean, and premature). Samples were was inoculated into Methyl Red Voges Proskauer (MR-VP
taken randomly, i.e. feces from 2 normal, 2 cesar, and 1 medium, and then was incubated for 2x24 hours at 300C. A
premature birth process. The data was analyzed with total of 3 drops of 40% KOH and 2 drops of alpha-naphthol
descriptive analysis. Determination of Bifidobacterium spp. reagent were added to the medium. A positive result was
was based on the morphological, chemical, and sugar test. interpreted as color change into the pink medium.
Manual characterization refers to Bergey's Determinative Oxidase test. One loop of the single colony was
Bacteriology, Cowan and Steel's Manual for the covered with a piece of filter paper, and then was added by
Identification of Medical Bacteria, World Journal of Dairy 1-2 drops of reagent (tetramethyl-D-phenylenediamine
& Food Sciences, International Journal of Food dihydrochloride). Positive result was interpreted with a
Microbiology, Applied and Environmental Microbiology, color change to maroon blue.
and followed by API 20A(Biomériux) test Measurement of Total Lactic Acid. A total of 2 mL of
24 hours bacterial culture was taken, and then was diluted
Procedures with 20 ml of distilled water and was shaken. Then, 3 drops
Isolation of phenolphthalein (pp 1%) were added and titrated with
Isolation of Bifidobacterium sp. from infants’ feces 0.1 N NaOH solution until a light pink color formation.
used the Modification of Gronlund (Modification of Resistance test to Lysozyme. MRSA medium was
Gronlund et al., 2009). Samples were taken aseptically added with the white part of the egg containing lysozyme
from 5 feces of under one month-aged infant with the (200 mg.ml-1). The medium was poured into a sterile dish
different birth process (normal, cesarean, and premature). and allowed to solidify. A 1 mL of bacterial culture was
A 5 g of feces was added with 45 mL of sterile distilled inoculated on Bifidobacterium medium, then was
water, then homogenized and diluted for up to 10-4. The anaerobically incubated for 24 hours at 35-360C. The
last two dilutions were used to isolate the bacteria using de bacterial resistance was shown by the growth of
Man, Rogosa, Sharpe Agar (MRSA) Oxoid with a spread Bifidobacterium in the medium
plate method in duplicate and were anaerobically incubated The pH range Growth test. Strains isolated MRS
at 300C for 24 hours. broth was grown in media with pH 4,7,9 and was incubated
for 24 hours at a temperature of 370C.
Identification
Morphological characterization. Morphology of API 20A (Biomériux) test
colony. Colony characterization was performed in MRSA, The colony isolated from infants feces was re-cultured
such as shape, size, surface, elevation and edge formation on MRSA medium and anaerobically incubated for 24
of the colony. hours, then 3 loops of the colony was taken and identified
Cellular characterization. Gram staining. One loop of with kit of API 20A. Indole test was added with mineral
the single colony was taken, and then was smeared on a and anaerobically incubated for 24 hours. Indole test
glass object. Crystal violet dye was dropped for 1 minute, interpretation needs the addition of XYL and EHR reagent.
and then was washed. Mordant solution (Lugol's iodine) The sugar test, glucose-arabinose, and glycerol-trehalose
was dropped as the second dye for one minute, and then need the addition of BCP reagent. Catalase test was added
was washed. Ethanol 96% was dropped gently, until it was by H2O2. The data was analyzed in the web of API 20
lightly crisp, and then was washed. Safranin was dropped
for one minute, and then was washed. The glass object was Inhibition test against pathogenic bacteria
air-dried, then was examined under the microscope. The Bacterial culture. Salmonella typhimurium and
cells of Gram-positive bacteria were purple in color. Escherichia coli were re-cultured in 100 mL Nutrient Broth
Meanwhile, Gram-negative bacteria were red. Motility (NB), and incubated in the shaker of incubator at 150
test. An ose of the single colony was stab inoculated on rotary per minute (rpm) for 24 hours.
 HENDRATI et al. – Bifidobacteria from infant faeces in Purwokerto, Indonesia 1267

Antibacterial activity. Suspension of S. typhimurium result in VP tests, because the bacteria do not ferment
and E. coli were inoculated on 10 mL Nutrient Agar (NA) mixed acid or butanediol fermentation. Oxidase test
medium (spread plate method). 0.1 mL supernatant was showed the positive result, indicated by blue-black color
dropped on Sterile paper disc with the diameter of 6 mm. formation after a few drops of tetramethyl-D-
Then, the paper disc was placed on the surface of NA phenylenediamine dihydrochloride. The color change is
medium (which had been inoculated with S. typhimurium due to the cytochrome enzyme oxidase the reagent.
and E. coli), then incubated for 24 hours at 300C. According to Feliatra et al. (2004), Bifidobacterium shows
Antimicrobial 1% of 24 hours culture of Bifidobacterium a positive result in oxidase test. Our data of lactic acid
spp. was inoculated into MRSB medium, and centrifuged at concentration from 35 isolates were ranged from 0.05-
13,000 rpm for 15 minutes. The supernatant was adjusted 0.224%. According to Silalahi and Ikhsan (2010), longer
with 1 N of NaOH to pH 6.5(neutralized), and heated at fermentation leads to the greater production of lactic acid.
1000C for 15 minutes. Antimicrobial activity was This is due to the increase length of fermentation time that
determined by measuring the clear zone around the paper increases the number of cell, therefore it increases the
disc after incubation. microbial activity to produce lactic acid.
Lysozyme test results showed that all isolates were able
to grow on de Man, Rogosa and Sharpe Agar (MRSA)
RESULTS AND DISCUSSION medium supplemented with the white part of the egg
(containing lysozyme). According to Gagnon et al. (2004),
Characterization test the Bifidobacterium is more resistant to white-egg
Based on the results of the morphological and lysozyme or digestive tract than other bacteria, even at the
biochemical test, there are 35 isolates belonging to the concentration of 300μg/ ml which is higher than the
genus Bifidobacterium. Morphology of colonies is similar concentration of intestinal lysozyme. The test results
in color, shape, surface and elevation, but quite different on showed that the pH range for the better growth of
the size and edge formation. The milky white colonies have Bifidobacterium spp. is at pH 7 than pH 4 and pH 9.
a flat edge/ smooth (entire), some are uneven like wool, According to Biavati et al. (2000), the Bifidobacterium is
round shape, shiny surface, convex elevation with varying an acid-tolerant microorganism with an optimum pH range
sizes. These isolates have morphological characters of 6.5-7.0
referring to genus Bifidobacterium , i.e. colony with milky
white color or slightly creamy, rounded form with the API 20A test
diameter of 0.1-0.5 mm as well as Gram-positive Indole test, after the addition of XYL reagent (2-3 min)
(Wasilewska and Bielecka 2003; Lievin 2000; Hadadji et and EHR (5 min), indicated a positive result with red color
al. 2005). and the negative result with yellow color. Urease test, after
the addition of XYL reagent (2-3 min) and EHR (5 min),
Biochemical tests indicated a positive result with red color and the negative
The results of biochemical tests of 35 isolates by Gram result with yellow color. The test of Glucose, mannitol,
staining test were confirmed as Gram-positive bacteria as lactose, saccharose, maltose, salicine, xylose, arabinose,
the purple cell. Gram-positive bacteria do not undergo glycerol, cellobiose, mannose, melezitose, raffinose,
decolorization so it stays shiny purple with color of crystal sorbitol, rhamnose, and trehalose, after the addition of BCP
violet staining and the end is not stained by safranin and reagent, showed that the positive result was indicated by
Gram-positive bacteria contains a peptidoglycan thick. yellow or yellow-green, and the negative result was
Motility test showed that all isolates showed no spreading indicated by purple color. Gelatin positive test was
pellicle formation on the surface of the medium and indicated by the occurrence of black pigment with diffusion
without turbidity;therefore it was interpreted as nonmotile and negative result was without diffusion. Esulin positive
bacteria. Garrity et al. (2005) showed that the motility test test was shown in black brown and fluorescent, the
positive if growth isolates form a pellicle widespreading on negative result was shown in yellow without fluorescent.
the surface of the medium, while it didn’t show the spread The result of API test showed that 17 of 35 isolates were
of pellicle on the medium surface, named nonmotile. The confirmed as genus Bifidobacterium namely BC4, BC5,
positive reaction of catalase test is indicated by the BC6, BC7, BC8, BC10, BC13, BC14, BC15, BC16, BC17,
formation of O2 gas bubbles as the breakdown of H2O2 by BC18, BC19, BP1, BP6, BP7, dan BP9 (Figure 1). In
catalase enzyme produced by the bacteria. The negative normal birth process, contact between infant and
reaction to calatase test is indicated by no formation of gas nonpathogenic bacteria in mother’s vagina bacteria lead to
bubbles. According to Hadadji et al.(2005), colonization of probiotic bacteria, while in caesar and
Bifidobacterium is catalase negative-bacteria. Indole test premature birth processes, infant doesn’t have a chance of
showed a negative result, indicated by no formation of the that contact, therefore colonization of probiotic bacteria
pink ring on the surface of the medium. Zinedine and Faid doesn't occur. In this research, the feces of infants in
(2007) stated that Bifidobacterium is negative in indole normal birth process did not contain any Bifidobacteria in
test.Voges-Proskauer test showed the negative result, their fecal samples. This is presumably due to fecal
indicated by color change into pink or dark red after a few samples of infants having mothers who live in rural areas
drops of 40% KOH and alpha-naphthol 5%. Venkatesn et that their modes of feeding do not fulfill the nutrition
al (2012) stated that Bifidobacterium shows a negative during pregnancy (Figure 1, 2 and 3).
1268 B I O D I V E R S I T A S 18 (3): 1265-1269, July 2017

Discussion
The results showed that the diameters of the inhibitory
zone of supernatant without NaOH 1 N addition are varied.
This result is due to the present of lactic acid as the
antimicrobial agent. The present of Lactic acid from the
fermentation of the Bifidobacterium spp. has several
advantages, such as high solubility, low toxicity and as an
effective antibacterial property (Lye et al.2009). The lactic
acid can suppress the growth of pathogenic bacteria, and
reduce the infection and carcinogenic effects (Venkatesan
et al. 2012).
Figure 1. Distribution of 35 isolates suspected of Bifidobacterium The clear zone formation indicates the ability of
after API 20A test. Note: Nnb: normal birth, non Bifidobacterium, metabolite of Bifidobacterium spp. in inhibiting E. coli and
Nbi: normal birth, Bifidobacterium, Cnb: Caesarean birth, non
Bifidobacterium, Cbi: Caesarean birth, Bifidobacterium, Pnb:
S. typhimurium. The NaOH addition to pH 6.5 aims to
Premature birth, non Bifidobacterium, Pbi: Premature birth, eliminate the influence of the produced acid during
Bifidobacterium metabolism, therefore the inhibition of E. coli and S.
typhimurium is expected due to the activity of bacteriocins.
Supernatant consisting of bacteriocins produces a clear
Inhibitory activity test zone with clear boundaries because bacteriocins are
Figure 2 and Figure 3 show that the inhibitory activity bactericidal which produce acid or other antimicrobial
test of 17 isolates belonging into Bifidobacterium spp. components which are bacteriostatic. According to Ray and
against E. coli and S. typhimurium. The diameter of the Field (1992), bacteriocins produce a really clear zone. If
inhibitory zone of supernatant with NaOH 1 N addition there is no clear zone, it is estimated as the action of
against E. coli ranges from 6.00-13.8 mm and diameter of hydrogen peroxide, acid or diacetyl. Hydrogen peroxide is
the inhibitory zone of supernatant without the addition of produced by lactic acid bacteria that can damage the
NaOH 1 N against E. coli ranges from 6.8-12.5 mm. The structure of the microbial lipid membrane, thus will
diameter of the inhibitory zone of supernatant with NaOH increase the membrane permeability, and then it will
1 N addition against S. typhimurium ranges from 6.00-11.5 damage the structure of nucleic acids and proteins of the
mm and diameter of the inhibitory zone of supernatant cell (De Vuyst and Leroy 2007).
without the addition of NaOH 1 N against S. typhimurium Bacteriocin is secondary metabolites of lactic acid
ranges from 7.19-12 mm. bacteria. Bacteriocins have the same way of working with
antibiotic, which is able to inhibit the growth of certain
bacteria. Bacteriocins in performing the antimicrobial
activity will attack the cytoplasm (Naidu and Clemens,
2000). Mechanism of bactericidal activity of bacteriocins
generally starts from a molecule bacteriocins that come into
direct contact with the cell membrane, therefore the process
of this contact causes the disturbation of membrane
potential, i.e. destabilization depolarization of the
cytoplasmic membrane, therefore the cells cannot survive.
Membrane fluidity impacts the formation of holes or pores
in the cell membrane through the disruption of Proton
Motive Force (PMV) (Rodney et al. 2014).
In conclusion, there are 35 isolates expected to be
Figure 2. Diameter of inhibitory zone secondary metabolites of
Bifidobacterium without the addition of NaOH
Bifidobacterium group. After the API 20A test, 17 isolates
from normal birth process, 14 isolates from cesarean birth
process, and 3 isolates from premature were confirmed to
be genus Bifidobacterium spp.. All these isolates inhibited
the growth of S. typhimurium and E. coli and can also be
used as potential inoculum in fermented healthy beverages.

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