International Journal of Scientific and Research Publications, Volume 5, Issue 4, April 2015 1

ISSN 2250-3153

Isolation and Identification of lactobacilli from raw milk

Abstract- Milk and milk products have been used by human population since ancient ages and have been a well known source of
lactobacilli. The present study is directed towards the study of prevalence, isolation and identification of Lactobacillus species in milk.
Milk samples were collected from the cow and buffalo sheds of a local dairy in the Aarey Milk Colony which is a major supplier to
the city of Mumbai. A total of 40 milk samples were obtained and 163 colonies were isolated from them. These colonies were
subjected to characterization by standard microbiological methods. On the basis of physiological tests and sugar utilization pattern, all
the 163 isolates were confirmed to belong to the genus Lactobacillus: L.fermentum (48%), L.acidophilus (34%), L.viridescens (8%),
L.brevis (5%), L.gasseri (4%) whereas two isolates could not be identified upto the species level. The results indicate that
L.fermentum is predominant in the milk obtained from this sector.

Index Terms- Characterization; Isolation; Identification; Lactobacillus fermentum; Milk samples

I. INTRODUCTION
The genus Lactobacillus consists of a genetically and physiologically diverse group of rod-shaped, Gram-positive, non-spore forming,
non-pigmented, catalase negative and microaerophilic to strictly anaerobic organisms.7Among lactic acid bacteria (LAB), lactobacilli
present a diverse group of homofermentative and heterofermentative species which can produce a variety of substances such as lactic
acid, ethanol, formic acid, acetone, hydrogen peroxide, diacetyl etc.15 Additionally they also produce a variety of exopolysaccharides
(EPS). The EPS vary in composition, structure and size with the producing organism. Although tasteless, the EPS from LAB increases
the residence time the milk products spend in the mouth and hence, imparts an enhanced perception of taste. 1 Additionally,
exopolysaccharides of microbial origin are gaining importance because of their potential applications in food industries as texturizers,
viscosifiers, emulsifiers and syneresis-lowering agents, for their pseudoplastic rheological behavior and water binding capacity. 1, 6

Lactobacilli possess two major advantages in that they are GRAS and are probiotics 2. The LAB are widely used in manufacturing
fermented food products and can also be safely used for medical and veterinary applications 3.

Lactic acid bacteria (LAB) and physiologically related group of gram-positive bacteria have also been known to produce a variety of
antimicrobials called bacteriocins. Bacteriocins are generally defined as extracellular peptides or proteins exhibiting bactericidal
activity against species closely related species or strains. Although bacteriocins may be found in many Gram positive and Gram-
negative bacteria, those produced by LAB have received particular attention in recent years due to their potential application in the
food industry as natural preservatives 4. Lactic acid bacteria and their metabolites have been shown to play an important role in
improving microbiological quality and shelf life of many fermented food products and provide a good example of bio preservation 5.

In the food industry, LAB is widely used as starter cultures and has been cited to be part of human microbiota. In raw milk and dairy
products such as cheeses, yoghurts and fermented milks, lactobacilli are naturally present or added intentionally, for technological
reasons or to generate a health benefit for the consumer as they strongly determine the flavor, texture and the nutritional value of the
food and feed products.

From the health point of view, ingestion of live cells of certain strains of lactobacilli in adequate amounts is believed to confer several
beneficial physiological effects on the host amongst these is the maintenance of a healthy and equilibrated intestinal flora and
enhanced resistance to intestinal infections.7 Due to the immense amount of varied products and their diversified applications, LAB
pose as an avenue which can be exploited with both health and economic benefits.

The present study deals with the isolation and identification of lactobacilli from various milk samples obtained from Cows and
Buffaloes harboured in the sheds of the Aarey Milk Colony and which provide an important channel of milk supply throughout the
eastern and the western suburbs of Mumbai.

www.ijsrp.org
International Journal of Scientific and Research Publications, Volume 5, Issue 4, April 2015 2
ISSN 2250-3153

II. MATERIALS AND METHODS

Milk samples were collected in sterile containers at the sheds and were transported to the lab in temperature controlled boxes. Further
analyses of the samples were carried out at the lab.

ISOLATION AND IDENTIFICATION OF LACTOBACILLUS

Appropriate dilutions of the collected milk samples were made in normal saline and pour plated on MRS agar and incubated at 37°C
anaerobically for 24 to 48 hours9. At the end of 48 hours, when the colonies became predominant, morphologically distinct and well
isolated colonies were picked and transferred to new MRS agar plates by streaking. Colonies showing typical characteristics of
lactobacilli on agar surface were picked up randomly and transferred into MRS broth for further enrichment. Further, their purity was
checked on MRS agar.

The pure isolates were subjected to identification as per the Bergey’s Manual of Determinative bacteriology and open source
softwares named PIBWin and IDENTAX. Macroscopic appearance of all the colonies was examined for cultural and morphological
characteristics. Size, shape, colour and texture of the colonies were recorded.

The isolates were stained by Gram's method and examined under microscope for purity and those isolates readily identified as Gram
positive rods and catalase negative were included for further characterization 13 which included cytochrome oxidase; growth at 15°C
and 45°C; acid production from carbohydrates (1 % w/v) - L-arabinose, D-fructose, cellobiose, , esculin, lactose, maltose, D-
galactose, melezitose, melebiose, mannitol, D-mannose, raffinose, rhamnose, D-ribose, salicin, sorbitol, sucrose, trehalose and D-
xylose in MRS broth devoid of glucose and beef extract with phenol red as indicator; production of acid and gas from 1 % glucose
(MRS broth without beef extract); methyl red and Voges-Proskauer test in MRVP medium; production of ammonia from arginine;
nitrate reduction in nitrate broth; indole production in tryptone broth and growth on acetate agar. 10,11,12

MEDIA

The strains were maintained on MRS medium with the following composition: peptone 1%, yeast extract 0.5%, beef extract 0.5%,
glucose 2%, dipotassium phosphate 0.2%, dibasic ammonium citrate 0.2%, sodium acetate 0.5%, magnesium sulphate 0.01%,
manganese sulphate 0.005%, Tween 80 (0.1%) and agar 3% (for solid medium).

Catalase test was performed with the help of hydrogen peroxide.(3% H 2O2 reagent grade).

Carbohydrate utilization was checked in 1% (w/v) MRS broth containing the specific sugar and devoid of glucose and beef extract
containing phenol red as indicator

Acid and gas production from glucose was checked with the help of inverted Durham’s tube containing 1% (w/v) glucose in MRS
broth without beef extract.

MR-VP tests were performed in MRVP broth. For MR test, methyl red was used as a reagent whereas for VP test alpha naphthol and
KOH were used as reagents.

Nitrate test was performed in Nitrate broth and tested with alpha- naphthylamine and sulphanilic acid.

Indole test was performed in tryptone broth and tested with the help of Kovac’s reagent.

Citrate test was performed on Simon’s Citrate agar.

www.ijsrp.org
International Journal of Scientific and Research Publications, Volume 5, Issue 4, April 2015 3
ISSN 2250-3153

III. RESULTS

IDENTIFICATION

The preliminary investigations included macroscopic analysis, microscopic analysis (Gram-positive bacilli), lactic acid biosynthesis,
endospore test, milk coagulation activities and the negative catalase reaction permitted the classification of the working bacterium into
the Lactobacillus genus. Microscopically they were Gram-positive rod shaped, non- motile, catalase negative and absence of
endospore. The isolates were tolerant to a range of salt concentrations (1-9%) and also have the ability to coagulate milk. All these key
characteristics helped to classify the isolates as lactobacilli.

BIOCHEMICAL TESTS

Identification upto the species level was carried out with the help of biochemical tests given in Bergey’s Manual of Determinative
bacteriology whereas open source softwares like IDENTAX and PIBWin were used to aid the identification and help develop an
identification scheme.8,11,14(Table 1)

Table 1: Scheme used for the identification of lactobacilli8

www.ijsrp.org
International Journal of Scientific and Research Publications, Volume 5, Issue 4, April 2015 4
ISSN 2250-3153

IV.DISCUSSION

Milk and milk products have always been an important source exhibiting a plethora of lactobacilli. A vast majority of efforts are
channelized on isolating lactobacilli and exploiting them for health related benefits. The present study was aimed at isolating
lactobacilli from raw milk samples obtained from a local dairy in the Aarey Milk Colony which is an important milk supplier to the
city of Mumbai. A total of 163 isolates were isolated and identified from the 40 milk samples obtained from the dairy. These isolates
were maintained on MRS medium and subcultured as required. After confirming the purity of the isolates, they were subjected to
microscopic and biochemical analysis. Those isolates which appeared to be rod-shaped, Gram-positive, non-spore forming, non-
pigmented, catalase negative and microaerophilic to strictly anaerobic, were confirmed to belong to the genus lactobacillus. These
isolates were further subjected to biochemical analysis and sugar fermentation patterns according to the Bergey’s Manual of
Determinative bacteriology and also with open source softwares like IDENTAX and PIBWin. Out of the 163 isolates obtained, 78
were found to belong to L.fermentum, 56 were found to belong to L.acidophilus, 12 were found to belong to L.viridescens,8 were
found to belong to L.brevis, 7 were found to belong to L.gasseri whereas two isolates could not be identified upto the species level.
These results indicate that L.fermentum is predominant in milk obtained from this sector followed by L.acidophilus(Figure1).

Figure 1: Percentage of lactobacillus species amongst the isolates.

V.CONCLUSION

The results of this study indicate that cow and buffalo milk exhibit a wide diversity of lactobacilli occurring naturally in the milk and
can be used as a potential natural source to isolate a variety of strains of lactobacilli. According to the current study, the results
indicate that L.fermentum is predominant in milk obtained from this sector of the Aarey Milk Colony and may play an important role
in the quality of the milk. Since some strains of lactobacilli possess potential probiotic and therapeutic properties including anti-
inflammatory and anti-cancer activities,16,17,18 as well as other features of interest, these isolates can be further screened for their
probiotic and related properties and exploited for health and economic benefits.

VI.REFERENCES

[1]. Vimlesh Yadav, Siddalingaiya Gurudutt Prappulla, Alok Jha, Amrita Poonia. A novel exopolysaccharide from probiotic Lactobacillus fermentum CFR 2195:
Production, purification and characterization. Biotechnology, Bioinformatics and Bioengineering 2011,pp 415-421

[2]. Mahantesh M Patil, Ajay Pal, T. Anand and K V Ramana. Isolation and Identification of lactic acid bacteria from curd and cucumber. Indian Journal of
Biotechnology Volume 9,2010, pp 166-172

[3]. Djomne Victor SIELADIE; Ngoufack François ZAMBOU1; Pierre Marie KAKTCHAM;Alberto CRESCI and Florence FONTEH. Probiotic properties of
lactobacilli strains isolated from raw cow milk in the western highlands of Cameroon. Innovative Romanian Food Biotechnology Vol. 9, Issue of September, 2011.

www.ijsrp.org
International Journal of Scientific and Research Publications, Volume 5, Issue 4, April 2015 5
ISSN 2250-3153

[4]. Khosro Issazadeh, Mohammad reza majid - Khoshkholgh Pahlaviani and Alireza Massiha. Isolation of Lactobacillus Species from Sediments of Caspian Sea for
Bacteriocin Production. 2nd International Conference on Biomedical Engineering and Technology IPCBEE vol. 34, 2012

[5].V.O. Adetunj and G.O. Adegok. Bacteriocin and cellulose production by lactic acid bacteria isolated from West African soft cheese. African Journal of
Biotechnology. 6,2007, 2616-2619

[6]. Kodali VP, Das S, Sen R.An exopolysaccharide from a Probiotic: Biosynthesis dynamics, Composition and Emulsifying Activity. Food Res. Int., 42, 2009, 695-699

[7]. M.Z. Hoque, F. Akter, K.M. Hossain, M.S.M. Rahman, M.M. Billah and K.M.D. Islam. Isolation, Identification and Analysis of Probiotic Properties of Lactobacillus
Spp. From Selective Regional Yoghurts. World Journal of Dairy & Food Sciences 5 (1),2010, 39-46

[8]. Parvathy Seema Nair and Puthuvallil Kumaran Surendran. Biochemical characterization of lactic acid bacteria isolated from fish and prawn. Journal of culture
collections Volume 4,2004-2005, pp. 48-52

[9]. Ramalingam, C. and Anvita Karara. Isolation and characterization of bacteriocin producing lactobacillus and bacteriocin from traditional fermented food.
International Journal of Microbiology and Bioinformatics - Vol.1, Issue, 1, November, 2011, pp.010-015

[10]. Aarti Bhardwaj, Monica Puniya, K. P. S. Sangu, Sanjay Kumar and Tejpal Dhewa. Isolation and Biochemical Characterization of Lactobacillus species Isolated
from Dahi. A Journal of Dairy Science and Technology Volume 1, Issue 2, August, 2012, pp18-31

[11]. Ghanbari M, Rezaei M, Jami M and Nazari, R. M. Isolation and characterization of Lactobacillus species from intestinal contents of beluga (Huso huso) and
Persian sturgeon (Acipenser persicus). Iranian Journal of Veterinary Research, Shiraz University, Vol. 10, No. 2, Ser. No. 27,2009

[12]. Vamanu Emanuel, Vamanu Adrian, Popa Ovidiu and Câmpeanu Gheorghe. Isolation of a Lactobacillus plantarum strain used for obtaining a product for the
preservation of fodders. African Journal of Biotechnology Vol. 4 (5), May 2005, pp. 403-408

[13]. Anandharaj.M*, Sivasankari.B. Isolation of potential probiotic Lactobacillus strains from human milk. International Journal of Research in Pharmacy and Life
Sciences, Vol. 1(1): 2013, 26-29

[14]. Özlem Erdogrul, Feryal Erbilir. Isolation and Characterization of Lactobacillus bulgaricus and Lactobacillus casei from Various Foods. Turkish Journal of
Biology ,2006, 39-44

[15]. P. Lonkar, S. D. Harne, D. R. Kalorey and N. V. Kurkure. Isolation, In vitro Antibacterial Activity, Bacterial Sensitivity and Plasmid Profile of Lactobacilli.
Asian-Australian Journal of Animal Sciences. Vol 18, No. 9 ,2005, 1336-1342

[16]. Azizah Ahma d Fauzi, Zaleha Shafi ei , Badiah Baha rin & Nurulhuda Mohd. Isolation of Lactobacillus from Periodontally Healthy Subjects and its Antimicrobial
Activity against Periodontal Pathogens. Sains Malaysiana 42(1).2013 ,19–24

[17]. Lanjar Sumarno, Djumali Mangunwidjaja , Anas M. Fauzi, Khaswar Syamsu, Nastiti Siswi Indrasti , Bambang Prasetya. Isolation and Fermentation of
Lactobacillus plantarum JR64 as an Omega 6 Probiotic Producer. International Journal of Science and Engineering Vol. 2(2):, Dec 2011 ,42-47

[18]. B. Nandhini and M. Palaniswamy. Anticancer effect of goat milk fermented by lactobacillus plantarum and lactobacillus paracasei. International Journal of
Pharmacy and Pharmaceutical Sciences Vol 5, 2013

AUTHORS
First Author – Sarangdhar Mithun, M.Sc, Ramnarain Ruia College, mithunsarangdhar@gmail.com
Second Author – Vora Dipak, Ph.D, Ramnarain Ruia College, dipakv55@gmail.com
Third Author – Sarang Sheela, M.Sc, Ramnarain Ruia College, sheelasarang@gmail.com

Correspondence Author – Sarangdhar Mithun, mithunsarangdhar@gmail.com , 9224385611

www.ijsrp.org