World Journal of Microbiology

Vol. 4(1), pp. 093-099, June, 2017. www.premierpublishers.org. ISSN: 2141-5032

WJM

Research Article

Isolation studies of Bacteriocin producing Lactic Acid

Bacteria from raw goat milk
Reshma Badi and Manish Bhat*
School of Biotechnology and Bioinformatics, D.Y. Patil University, Navi Mumbai. Plot no. 50, Sector-15, CBD Belapur,
Navi Mumbai, Pincode-400614, Maharashtra, India.

Probiotics are live microorganisms which when administered in appropriate amount gives health
benefit on the host. In the current study raw goat milk obtained from Belapur village (Belapur,
Navi Mumbai, Pin-400614, Maharashtra, India) was the source of isolated lactic acid bacteria. Out
of 118 screened isolates, only one isolate i.e. RL 76 showed antimicrobial activity upon primary
screening. Optimization of the isolate was carried out to check its bacteriocin producing potential.
Isolate grew well at 370C and pH 7. Isolate RL 76 tolerated 2% of bile salt and showed no
haemolytic activity. Upon morphological and biochemical tests, the isolate RL76 was identified
as Lactobacillus casei. The extracted cell free supernatant was partially purified by ammonium
sulphate precipitation. Partially purified cell free supernatant showed antibacterial activity against
Staphylococcus aureus, Streptococcus fecalis, Klebsiella pneumonia, Pseudomonas aeruginosa,
Salmonella typhi and Escherichia coli. The study indicates potential of the isolate as a good
probiotic species.

Keywords: Probiotics, Goat milk, Bacteriocin, antibacterial activity, bile salts.

INTRODUCTION

Lactic acid bacteria (LAB) have been studied worldwide for
their potentially antimicrobial bacteriocin. This bacteriocin
had been used so far as food preservative (Zendo T.,
2013). Gram positive bacteria have been found to produce
more effective bacteriocin in comparision to Gram
negative bacteria (Ingolf et al., 2007). LAB bacteriocin
studied so far has been found to show diversity with
respect to physiological ribosomal peptides and spectrum
of antimicrobial activity (Cintas L., 2001). Bacteriocin has
been classified into three groups as per their structure and
physicochemical properties. Some bacteriocins are non-
immunogenic, biodegradable and have been studied for
their anticancer effects (Kaur and Kaur, 2015). Bacteriocin
from LAB not only finds applications in food industry but
also in medical sector. Bacteriocin has been shown to play
important role in in vivo gut interactions (Vuyst and Leroy,
2007). Antimicrobial activity of Lactobacillus species
isolated from raw goat milk against Staphylococcus aureus
was studied (Anas et al., 2008). Bacteriocin of
Isolation studies of Bacteriocin producing Lactic Acid Bacteria from raw goat milk
Lactobacillus brevis WD19 was reported from goat milk
with proteolytic activity (Dib et al., 2014). Acid tolerant
Bacteriocin extracted from Lactobacillus plantarumgoat
milk showed significant antimicrobial activity against
Staphylococcus aureus and Bacillus subtilis (Sridevi et al.,
2015). Probiotic Lactobacillus strains isolated from raw
goat milk showed inhibitory properties against Bacillus
subtilis and Pseudomonas aeruginosa (Barua et al., 2015).
Lactococcus lactis subsp. lactis DF4Mi a
bacteriocinogenic strain was isolated from goat milk, and
studied for its antimicrobial activity.
*Corresponding author: Dr. Manish R. Bhat, Assistant
Professor, SBB, DYPUNM., School of Biotechnology and
Bioinformatics, D.Y. Patil University, Navi Mumbai. Plot no.
50, Sector-15, CBD Belapur, Navi Mumbai, Pincode-
400614, Maharashtra, India Tel.: 91-22-27573600, Email-
manish.bhat@dypatil.edu,reshmabadi92@gmail.com
Badi and Bhat 094
It possesses broad spectrum of activity. Found sensitive to
proteolytic enzymes, resistant to heat and extreme pH
(Furtado et al., 2014). In the current study an attempt was
made to isolate bacteriocin producing Lactic acid bacterial
species from raw goat milk along with its optimization
studies and study of its antimicrobial potential against
pathogens.
MATERIALS AND METHODS
Sample collection and preparation
The raw goat milk sample was obtained from Belapur
village, Navi Mumbai, Maharashtra, India. Proper
sterilization of teats was carried out with sterile distilled
water. Then milk sample was collected in a sterile test
tube, transported and stored at 4C until further used. The
sample was then serially diluted and used for further
analysis (Perin et al., 2014)
Bacterial strains and media
Lactic Acid Bacteria were isolated from the samples by
plating appropriate dilutions on MRS (De Man J D, Rogosa
M and Sharpe M E) (MRSA) (Make- Hi media) agar plates
by spread plate method. Plates were incubated for 48
hours at 370C under anaerobic conditions (Perin et al.,
2014).
Primary screening of antimicrobial activity of isolates
All the isolates obtained on MRSA plates were used for
screening their antimicrobial potential. All isolates were
grown in sterile MRS broth and incubated at 370C for 24
hours. After incubation 10ml of the culture was centrifuged
at 10,000 rpm for 10 min at 40C. The cell free supernatant
was used for agar well diffusion method to check their
antimicrobial potential (Patil M., 2010). Staphylococcus
aureus and E. coli were used during agar well diffusion
method.
Optimization of growth parameters
Temperature and pH
Optimum temperature and pH were determined for the
isolate RL76 under study. Optimum growth temperature
was determined by growing the selected isolate in Sterile
MRS broth and incubating at different temperatures (20,
37 and 45C). Sterile MRS broth of different pH (pH 5,6,7,8
and 9) were inoculated and incubated to determine the
optimum pH. These parameters for highest growth of the
identified Lactobacillus spp. were determined by
measuring and comparing the optical density (OD) at 530
nm (Barua et al., 2015).
Isolation studies of Bacteriocin producing Lactic Acid Bacteria from raw goat milk
Bile salt tolerance of the isolate
Sterile 10 ml of MRS broth was inoculated with 0.5 ml of
varied concentration (i.e.; 0.1, 0.5, 1.0, 2.0 and 3.0% [w/v])
of bile salt. All the tubes were then inoculated with 0.1 ml
suspension of the isolate RL 76. The tubes were then
incubated at 37C for 24 hours. Presence of turbidity in
tubes was observed (Barua et al., 2015).
Hemolytic Activity
Suspension of the isolate was spot inoculated on blood
agar, containing 5% (w/v) sheep blood and incubated for
24h at 30C. Blood agar plates were examined for zone of
inhibition around the spots. Modified method was used
(Anas et al., 2008).
Identification of isolate
The isolate under study was gram stained and examined
microscopically for cellular morphology. The biochemical
tests included the IMViC tests and sugar fermentations
(MacFaddin J., 2000). . Biochemical characteristics were
studied with Himedia Kit (KB001HIIMViCTM Biochemical
Test Kit) (for gram positive organisms) with 24 hours old
suspension of the isolate (Vishwanathan et al.,2015).
Partial purification of cell free supernatant of potential
isolate
Out of total 118 isolates only one isolate i.e. RL76
exhibited antimicrobial action. Strain RL 76 showing
maximum anti-microbial activity was grown in MRS broth
at 370C for 24 hours. After incubation 10ml of the culture
was centrifuged at 10,000 rpm for 10 min at 40C. The cell
free supernatant was used as crude bacteriocin.
Antimicrobial activity of all the isolates was carried out by
agar well diffusion method (Patil M., 2010). The cell free
supernatant obtained from isolate RL76 was saturated
with 20%-60% ammonium sulphate, stirred on a magnetic
stirrer and stored at 40C overnight. The precipitate was
collected by centrifugation at 10,000 rpm for 30 min.
Partially purified cell free supernatant swas reconstituted
in double the volume of 20mM sodium phosphate buffer at
pH 6 and stored at 40C for further characterization (Ravi
SNV et al., 2012).
Antimicrobial assay of partially purified cell free
supernatant
2ml of Partially purified cell free supernatant was used for
study of antimicrobial assay..Agar well diffusion method
was used for antimicrobial assay (Patil M.,2010) Test
microorganisms used were Staphylococcus aureus,
Streptococcus fecalis, Bacillus subtilis, Klebsiella
pneumoniae, Pseudomonas aeruginosa, Salmonella typhi
and E. coli.
 World J. Microbiol. 095

Table 1. Optimization of isolate RL 76.

Growth at different temperatures at 200C 370C 450C

- +++ +
Growth at different pH 5.0 6.0 7.0 8.0 9.0
- - +++ + -
Growth at different bile salt concentration in % 0.1 0.5 1.0 2.0 3.0

++++ +++ +++ +++ -

Key: + indicates positive growth, - indicate negative growth

Table 2. Colony Characteristics of the isolate RL 76

Characteristics Observation

Size 2.2mm

Shape Circular
Elevation Sphere
Margin Regular
Opacity Translucent
Consistency sticky
Pigmentation Off white/creamy

RESULTS concluded that the isolate under study could be

In the current study LAB especially Lactobacillus spp. from
goat milk samples of village Belapur, Navi Mumbai,
Maharashtra, India was undertaken.
Primary screening was carried out with obtained isolates
on MRSA plates by agar well diffusion method. Out of total
screened 118 isolates only one isolate inhibited the growth
of Staphylococcus aureus and Salmonella typhi. Other
isolates under study did not show antimicrobial activity
against used microorganism. Results are not shown.
The isolate under study exhibited heavy growth at 37 0C
and tolerated pH of 7. Isolate RL76 found growing up to 2
% of bile salt concentration (Table 1).
Lactobacillus casei.
Partially purified cell free supernatant of isolate RL 76 was
found effective against two Gram positive bacterias
namely, Staphylococcus aureus and Streptococcus fecalis
(Figure 1 and 2) and three Gram negative organisms
namely Klebsiella pneumonia, Pseudomonas aeruginosa,
E. coli and Salmonella typhi (Figure 3,4,5,6). Zone of
inhibition of isolate RL 76 against pathogens used are
shown in Table 4.
DISCUSSION

Isolate did not exhibit any haemolytic activity on blood agar
plate. Morphological studies were on sterile MRSA. Colony
characteristics were studied. The isolate was found
showing sticky, circular, cream colored, opaque colonies
with with entire margin and convex elevation. Upon Gram
staining the isolate was found to Gram positive bacilli
shaped structure. (Table 2).
Biochemical characteristics were studied with the help of
Himedia Kit (KB001HIIMViCTM Biochemical Test Kit) (for
gram positive cocci/bacilli organisms). Results of
biochemical characteristics are as per Table 3.
Morphological and biochemical results of the isolate RL76
were compared with Bergey`s Manual of Determinative
Bacteriology for gram positive bacteria. It can be
Isolation studies of Bacteriocin producing Lactic Acid Bacteria from raw goat milk
Lactic acid bacteria have been found effective on various
concerns of human gastro intestinal tract like correction of
lactose intolerance, drug induced diarrhea and
inflammatory bowel syndrome. LAB and its metabolites
are used as probiotic and preservatives respectively. They
are considered as safe for human body (Zacharof and
Lovitt, 2012). Lactic acid bacteria produce various
compounds such as organic acids, diacetyl, hydrogen
peroxide, and bacteriocins during lactic acid
fermentations. Bacteriocins are basically antimicrobial
peptides becoming popular as therapeutics and food
preservatives (Yusuf and Hamid, 2013).
In the current study isolation of lactic acid bacteria (LAB)
from goat milk samples of village Belapur, Navi Mumbai,
Maharashtra, India was undertaken. MRS agar medium
was used for cultivation of Lactobacillus sp. during the
Badi and Bhat 096

Table 3. Biochemical characteristics of the isolate RL 76

Biochemical test Observed Result

Malonate -

Methyl Red -
Voges Proskauers -
Citrate +
ONPG -
Nitrate reductase +
Catalase -
Arginine -
Sucrose -
Mannitol +
Glucose +
Arabinose -
Trehalose +

Table 4. Diameter of zone of inhibition by isolate RL 76 against used pathogens

Organisms Diameter of zone of inhibition

(centimeters)
Staphylococcus aureus 2.0
Streptococcus fecalis 2.6
Pseudomonas aeruginosa 1.7
Salmonella typhi 2.5
Escherichia coli 1.9
Klebsiella pneumoniae 1.6

study. In total 118 isolates were obtained on MRSA plates.
Out of which apart from one of the isolate none other
exhibited antimicrobial activity upon primary screening.
Only isolate RL76 showed inhibition of used test
organisms. Hence only RL76 was selected for further
study (Jamaly et al., 2011).
Production of bacteriocin from Lactic acid bacteria is
generally influenced by various factors like temperature,
pH, incubation period (Saumya et al., 2012, Vasudevan et
al., 2014). In the study it was observed that isolate RL76
exhibited growth at temperature of 37 0C and tolerated pH
of 7. This was found in accordance with the cited
references.
Bile salt tolerance is one of the prerequisite for any species
of Lactic acid bacteria when it is used as probiotics (Begley
et al.,2004). Isolate under study tolerated 2% of bile salt.
Absence of haemolytic activity by probiotic strain indicates
that the strain is not virulent and safe for use as food (Vuyst
and Leroy 2007). Isolate RL 76 did not show any
haemolysis on blood agar plates. Thus it can be stated that
isolate under study is not virulent and it is in accordance
with reference cited.
On comparison of the morphological and biochemical
characteristics with Bergey`s Manual of Determinative
Bacteriology for gram positive bacteria it can be concluded
that the isolate under study (RL76) could be Lactobacillus
casei. Its further confirmation shall be carried out by 16S
rna sequencing method.
Isolation studies of Bacteriocin producing Lactic Acid Bacteria from raw goat milk
Antibacterial activity of bacteriocin producing Lactobacillus
isolated from raw cattle milk sample inhibited growth of
Bacillus mycoides, Staphylococcus aureus, Streptococcus
faecalis, Klebsiella pnuemoniae and Proteus vulgaris
(Mohankumar 2011). Bacteriocin isolated from goat milk
inhibited growth of Staphylococcus aureus (Mami et al.,
2011). Lactococcus and Lactobacillus Species isolated
from raw Goat milk sample found effective against growth
of Listeria monocytogenes ATCC 74902, Enterococcus
faecalis ATCC 29212, Escherichia coli and Pseudomonas
aeruginosa strains. Stability of bacteriocin was found
effective at pH 8-10 and athigher temperature (Mitjans et
al., 2016). L. bulgaricus Y34, Lactococcus lactis N22 and
S. thermophilus N27 isolated from fermented milk product
exhibited broad spectrum of inhibition (Mohamad and Ijah,
2013).
In the pilot screening out of 118 isolates only one i.e. RL76
exhibited broad spectrum of antimicrobial effect. Hence its
cell free extract was selected for further purification. The
cell free supernatant was found effective against two gram
positive bacteria namely, Staphylococcus aureus and
Streptococcus fecalis (Figure 1) and four gram negative
organisms namely Klebsiella pneumonia, Pseudomonas
aeruginosa, Salmonella typhi and Escherichia coli (Figure
2,3,4).
This differential spectrum of antimicrobial activity exhibited
by the isolate suggests that distinct inhibitory
 World J. Microbiol. 097

Figure 1 Antibacterial assay of cell free supernatant by agar well

diffusion method against Staphylococcus aureus.
Figure 4. Antibacterial assay of cell free supernatant by agar well
diffusion method against E.coli

component is present in the isolated species of LAB. This

is in accordance with studies carried out on LAB from food
products (Puttalingamma, 2006), (Assefa et al., 2008).
According to Vuyst, L (2007) antimicrobial activity may be
due to bioactive molecule producing such as ethanol,
formic acid, H2O2, diacetyletc of the probiotic strain. Thus
current study indicates that isolate RL 76 identified as
Lactobacillus casei and further identification can confirm
the genus and species level classification. However, as the
strain under study exhibited broad spectrum of
antibacterial activity substantiates that strain has potential
production ability of bacteriocin like antimicrobial
substances. Upon further study the detailed structure and
classified group of bacteriocin can be studied.
Figure 2. Antibacterial assay of cell free supernatant by agar well
diffusion method against Klebsiella pneumonia.
CONCLUSION

Lactic acid bacteria are widely used as probiotic all over

the world. Its bacteriocin is also being used as preservative
in many food-related products. There are many reports
which have substantiated goat milk as an alternative. The
current study also support that goat milk can be a good
alternative in the dairy market because of its bacteriocin.
Bacteriocin produced by isolate under study i.e.
Lactobacillus caseican be marketed upon further
purification and analysis.

ACKNOWLEDGEMENTS

The authors wish to thank, The Director, School of

Biotechnology and Bioinformatics, D Y Patil University,
Navi Mumbai, for providing the laboratory facilities and
Figure 3. Antibacterial assay of cell free supernatant by agar
well diffusion method against Pseudomonas aeruginosa. support for research endeavours of the faculty.

Isolation studies of Bacteriocin producing Lactic Acid Bacteria from raw goat milk
Badi and Bhat 098
REFERENCES
Anas M, Eddine HJ, Mebrouk K (2008). Antimicrobial
activity of Lactobacillus species isolated from algerian
raw goats milk against Staphylococcus aureus. World
Journal of Dairy & Food Sciences. 3(2): 39-49.
Assefa E, Beyene F and Santhanam A (2008). Isolation
and Characterization of inhibitory substance producing
Lactic Acid Bacteria from ErgonEthiopian traditional
fermented milk. Livestock Research for Rural
Development. 20(44).
Barua R, Abdullah HM, Masud AL, Md. NM, Mohammad
AH. (2015). Assessment of Potential Probiotic
Lactobacillus Strains Isolated from Goat Milk. IOSR
Journal of Pharmacy and Biological Sciences. 10(4):
09-15.
Begley, M., Guhan, CG and Hill C. (2004). Interaction
between bacteria and bile.FEMS Microbiol
Rev.29:625-651.
Cintas LM, Casaus MP, Herranz C, Nes IF and Hernnde
PE (2001). Review: Bacteriocins of Lactic acid
bacteria. Food Sci Tech Int .7(4):281305.
Dib W, Biscola V, Choiset Y (2014). Characterization of
a new isolate of Lactobacillus brevisWD19 from
Algerian goat milk with proteolytic activity. Global
Advanced Research Journal of Agricultural Science.
3(12): 423-432.
Furtado DN, Todorov SD, Landgraf M, Destro
MT, Franco BD (2014). Bacteriocinogenic Lactococcus
lactis subsp. lactis DF04Mi isolated from goat milk:
Characterization of the bacteriocin. Brazilian Journal of
Microbiology. 45(4): 1541-1550.
Ingolf FN, Yoon SS, DB (2007). Ribosomally synthesized
antimicrobial peptides (Bacteriocin) in Lactic acid
bacteria, A review, Food Sci. Biotechnol. 16 (5): 675-
690.
Jamaly N, Benjouad A, Bouksaim M (2011). Probiotic
potential of Lactobacillus strains isolated from known
popular traditional Moroccan dairy products. British
Microbiology Research Journal. 1(4): 79-94.
Kaur S and KaurS (2015). Bacteriocin as potential
anticancer agents. Front Pharmaco. 6: 1-11.
Larsen, AG, Vogensen, FK and Josephsen, J. (1993).
Antimicrobial activity of lactic acid bacteria isolated
from sour doughs: purification and characterization of
bavaricus M1401. J. Appl. Bacteriol. 75: 113-122.
MacFaddin JF (2000) Biochemical tests for Identification
of rd Medical Bacteria. Philadelphia: Lippincott
Williams and Wilkins (3): 363-367.
Mami A, Henni JE, Kihal M. (2008). Antimicrobial activity
of Lactobacillus Species isolated from Algerian raw
Goats milk against Staphylococcus aureus. World
Journal of Dairy & Food Sciences. 3(2): 39-49.
Mitjans NM,Coria MJ, Anzulovich AC,Risso GN,Castro
JM , Rezza IG, Stagnitta PV. (2016). Antimicrobial
activity of Lactococcus and Lactobacillus species
isolated from raw Goat milk. San Luis Argentina.
Isolation studies of Bacteriocin producing Lactic Acid Bacteria from raw goat milk
International Journal of Engineering Research &
Science. 2(8):141-147.
Mohammed, SSD and Ijah UJJ. (2013). Isolation and
screening of Lactic acid bacteria from fermented milk
products for bacteriocin production. Annals. Food
Science and Technology. 14(1): 122-128.
Mohankumar A (2011). Characterization and
antibacterial activity of bacteriocin producing
Lactobacillus isolated from raw cattle milk sample.
International Journal of Biology.3(3):128-143.
Patil MM, Pal A, Anand T and Ramana K V. (2010)
Isolation and Characterization of Lactic Acid Bacteria
from curd and cucumber. Indian Journal of
Biotechnology. 9: 130-135.
Perin LM and Nero LA (2014). Antagonistic Lactic acid
bacteria isolated from goat milk and identification of
novel nisin variant Lactococcus lactis. BMC
Microbiology.14(36): 1-9.
Puttalingamma V, Khyrunissa B and Bawa AS (2006).
Antimicrobial peptides new weapons against enteric
pathogens. Pakistan Journal of Nutrition. 5 (5): 432-
435.
Ravi SNV, Priyanka D, Reddy PS, Rajnikant P,
Kirankumar V and Indira M. (2012) Purification and
Characterization of Bacteriocin produced by isolated
from cow's milk. Int J. of Microbiol Res. 3(2): 132-137.
Soumya TV, John R, Jose Y (2012). Characterization of
Bacteriocin Produced by Lactobacillus sp and
Optimization of Cultural Conditions, International
Journal of Scientific and Research Publications. 2(12).
Sridevi V, Sirisha R, Swapna SN (2015). Screening of
probiotic Goat Milk and Cow Milk isolates for acid
resistance, antagonistic activity and tolerance to
antimicrobial activity of Spices: molecular identification
of potential probiotic goat milk isolate, G8. Int. J. Curr.
Microbiol. App. Sci. 4(8): 406-421.
Vasudevan S, Arulmoorthy MP, Ashokprabu V (2014).
Isolation, Identification and Characterization of
Bacteriocin from Lactobacillus lactis. BMR
Microbiology. 1(1): 1-18.
Viswanathan S,Preethi G, Veilumuthu P,Chairman K.
(2015). Goat milk Is it a good source of probiotics?.
Pharmaceutical and biological evaluations. 2 (4): 84-
90.
Vuyst LD and Leroy F. (2007). Bacteriocins from Lactic
Acid Bacteria: Production, Purification, and Food
Applications. J. MolMicrobiolBiotechnol.13:194199.
Yusuf MA (2013). Lactic Acid Bacteria: Bacteriocin
Producer: A Mini Review. IOSR Journal of Pharmacy.
3(4): 44-50.
Zacharof MP and Lovittb RW (2012). Bacteriocins
Produced by Lactic Acid Bacteria. APCBEE
Procedia.2:50 56.
Zendo T. (2013). Screening and characterization of novel
bacteriocin from Lactic acid bacteria, Biosci.
Biotechnol. Biochem. 77(5): 893-899.
 World J. Microbiol. 099

Accepted 8 February, 2017.

Citation: Badi R, Bhat M (2017). Isolation studies of
Bacteriocin producing Lactic Acid Bacteria from raw goat
milk. World Journal of Microbiology, 4(1): 064-071.

Copyright: 2017 Badi and Bhat. This is an open-access

article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
cited.

Isolation studies of Bacteriocin producing Lactic Acid Bacteria from raw goat milk