INT J CURR SCI 2011, 1: 109-115

RESEARCH ARTICLE

Production and antibacterial activity of bacteriocin by Lactobacillus paracasei isolated from

donkey milk
Ashokkumar Sa, R. Sree Krishnaa, V. Pavithrab, V. Hemalathab and Priya Ingalea*
a

Sachika, Institute for Training in Biomedical Technology, Gopalapuram, Chennai, India

Department of Microbiology, Adhiparasakthi College of Arts and Science, G.B.Nagar, Kalavai, Tamil Nadu, India
*Correspondence to: E-mail: pringale@gmail.com; Phone: 91-7829014014

Abstract
Bacteriocins obtained from lactic acid bacteria (LAB) in milk samples have found commercial applications in the food processing industry
as a biopreservative or probiotic. Donkey milk has recently found application as a safer substitute to human milk. Lactobacillus paracasei
was isolated from donkey milk. Crude bacteriocin was separated from the culture as cell-free supernatant. The crude bacteriocin was
subjected to antimicrobial activity against clinical pathogens. In the present study, Maximum inhibitory activity was observed against
Salmonella typhi followed by Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Minimum inhibitory concentration
(MIC) was also performed and the MIC index of 128 l was seen against Salmonella typhi. Staphylococcus aureus showed the least
susceptibility to the crude bacteriocin. The study suggests that the crude bacteriocin from Lactobacillus paracasei could be used as a
potential probiotic or a bio-preservative.
Keywords: donkey milk, bacteriocin, Lactobacillus paracasei, antimicrobial susceptibility, minimum inhibitory concentration (MIC)
Received: 09th October; Revised: 25th October; Accepted: 02ndNovember; IJCS New Liberty Group 2011
Introduction

hydrogen peroxide and reuterin during lactic Acid fermentations

Bacteriocins are a heterologous sub-group of ribosomally

(Gilliland and Speck, 1975; Holzapfel et al., 2001). Bacteriocin

synthesized antimicrobial peptides of bacterial origin that are

producing LAB have the generally recognized as safe (GRAS)

lethal to bacteria other than the producing strain (De vugst,

status and can be administrated to strengthen the barrier function

1994). In general, these moieties are cationic peptides that

of the commensals of the gut in animals and birds (Elisabetta

display hydrophobic or amphiphilic properties and the bacterial

Tome et al., 2008).

membrane is in most cases target for their activity. Depending

Milk represents a relevant source for the isolation of several

on the producer organism and classification criteria, bacteriocins

LAB strains. Donkey milk has recently stimulated scientific

can be classified into several groups as Class I, II and III (Jack

interest due to its attractive nutrient and functional contents.

and Jung, 2000). Some important bacteriocins include Nisin,

Due to its similarity in chemical composition to human milk, it

Diplococcin, Acidophilin, Bulgarican, Helveticins, Lactacins

is also considered a valid alternative for infants with severe IgE -

and Plantaricins (Nettles and Barefoot, 1993). Bacteriocins have

mediated cows milk protein allergy (Filomena et al., 2010,

potent antagonistic effect against important clinical pathogens as

Cavataio et al., 1996). Additionally, donkey milk is a strong

observed by (Ogunbanwo et al., 2003) and are extensively used

vasodilator, making it potentially useful in the prevention of

as potential antimicrobial agents in food preservatives.

atherosclerosis (Tafaro et al, 2007). It exerts an in vitro

Bacteriocins produced by LAB are predominantly present in

suppressing action against human lung tumors (Mao et al.,

carbohydrate rich environments such as food (dairy products

2009). Donkey milk is predominantly rich in whey proteins,

such as milk, curd, beverages), gastrointestinal tract of humans

such as lactoalbumin and lactoglobulin, which range from 35%

and animals and, in sewage and plant materials (Corsetti et al.,

to 50% of the total nitrogen fraction, compared to 20% in cows

1998). The genus Lactobacillus includes gram-positive rods with

milk. The total casein content is about 8.7 g/l, which is

a size range of 0.5-1.2 x 1-10 m and non-spore formers,

remarkably close to the amount found in human milk (Salimei

producing lactic acid as a fermented end-product (Aasen et al.,

et al., 2004). The present study was undertaken to isolate and

2000). Besides bacteriocin, LAB produces a variety of

investigate the antimicrobial activity of bacteriocin isolated from

antimicrobial compounds such as organic acid, diacetyl,

LAB in donkey milk against clinical isolates and purify

Ashokkumar et al., 2011

bacteriocin from the sample culture.

with various clinical manifestations.

Materials and methods

Screening for antibacterial susceptibility pattern

Isolation, media and culture conditions

The inhibitory activity of CFS and purified protein was screened

The donkey milk sample was collected in sterilized container

by Spot assay method (Sundaramoorthy and Sarvanan, 2010).

and stored at 4C till further use. No disease symptoms were

As per this protocol, 18 ml of Muller Hinton (MH, Hi-Media

observed in the source animal. During Isolation, 30 l of milk

Laboratory Pvt. Ltd. India) agar medium was prepared, sterilized

sample was inoculated on the De Man Rogosa Sharpe (MRs, Hi-

and 2 ml of CFS was mixed with the MH agar medium, poured

Media Laboratory Pvt. Ltd. India.) agar medium plates using

into sterile petri plates and allowed to solidify. The serially

spread plate technique. The plates were incubated anaerobically

diluted (10-1 to 10-9) pathogenic organisms were spotted on to

at

the plates, and were incubated at 37C for 24 hours and activity

37C

for

24-48

hours.

After

Incubation,

bacterial

identification tests were further performed.

was observed.

Identification

Minimum inhibitory concentration (MIC) assay

Based on the gram staining, cell morphology, growth at selected

The MIC is defined as the lowest concentration that completely

temperatures and standard biochemical tests, LAB species was

inhibits the growth for 24-hour culture (Thongson et al., 2004).

selected for further classification (Weiss, 1992). Further

MIC of CFS was determined by broth micro-dilution method as

identification of the species of the LAB isolate obtained was

recommended by National Committee for Clinical Laboratory

performed according to carbohydrate fermentation patterns and

Standards (NCCLS, 1997). Two-fold serial dilutions of the CFS

growth at 15C and 40C in MRS broth (Hi-Media Laboratory

ranging from 8 to 1024 l was added aseptically into the wells

Pvt. Ltd, India) as described in Bergeys Manual of Systematic

of Mueller Hinton (MH) broth tubes that were already seeded

Bacteriology (Garvie, 1986).

with the standardized inoculums (124 CFU/ml) of the test

Optimization assay

pathogenic bacteria. Sterile Dimethyl sulphoxide (DMSO)

The selected strain of L. paracasei was subjected to different

served as negative control. The tubes were incubated at 37C for

culture conditions to derive the optimum conditions for

24 hours. The lowest concentration of CFS showing a clear

bacteriocin production in MRS broth (Todorov and Dicks,

broth that reflects inhibition was considered as the MIC.

2004). Growth and bacteriocin production were estimated at

Partial purification of CFS

temperatures 20C, 30C, 40C, 50C and 60C, pH 4.0, 5.0,

The CFS extracted from L. paracasei was subjected to following

6.0, 7.0 and 8.0 and NaCl concentrations 0.5%, 1%, 1.5%, 2%,

partial purification methods.

2.5% and 3%.

a. Ammonium sulphate precipitation: A concentration of 60% of

Extraction of cell-free supernatant (CFS)

ammonium sulphate was added to 10 ml of CFS in sterile test

Following identification of Lactobacillus, the pure isolate was

tubes. It was allowed to precipitate for 24 hours at 4C. The

propagated in 1000 ml flask containing MRS broth (pH-6.0) and

mixture was then centrifuged at 10,000 rpm for 20 minutes and

was incubated at 37C for 72 hours anaerobically. A supernatant

the precipitate was re-suspended in 25 ml of 0.05 M potassium

that may contain crude bacteriocin, a cell-free solution was

phosphate buffer. The mixture was stirred at 4C for 24 hours.

obtained by centrifuging the culture at 10,000 rpm for 20

b. Dialysis: Following ammonium sulphate precipitation, the

minutes at 4C. After centrifugation the supernatant was

suspension was dialysed in a tubular cellulose membrane against

collected in a fresh sterile tube and pellets were discarded. The

2000 ml distilled water for 48 hours with 3 changes. After

CFS was adjusted to pH-6.0 using 1N NaOH. The amount of

dialysis, the purified sample was collected in sterile tubes. The

protein in the CFS was estimated by Lowrys method

amount of partially purified protein obtained from the dialysate

(Karthikeyan and Santosh, 2009).

was estimated by Lowry's method (Karthikeyan and Santosh,

Determination of antimicrobial susceptibility

2009).

Test pathogens to screen inhibitory activity of the protein (in

Purification of bacteriocin

CFS and purified protein fractionates) included Escherichia coli

Ion exchange chromatography using DEAE - Cellulose cation

(A), Klebsiella pneumoniae (B), Pseudomonas aeruginosa (C),

exchange column was selected for purification of bacteriocin

Salmonella typhi (D) and Staphylococcus aureus (E) associated

from the dialysate. The elution was performed by using a linear

Ashokkumar et al., 2011

gradient citrate phosphate buffer ranging from pH 2.6 to 7.0.

Table 1c. Sugar Fermentation Test

Protein content was determined by Lowrys method. All

procedures were performed in a cold room.

Sugar Fermentation Test

Result

Screening of purified bacteriocin activity

Arabinose

Negative

Based on the protein estimation by Lowrys method, selected

Mannose

Positive

tubes containing maximum purified protein were subjected to

Fructose

Positive

screening for bacteriocin content using spot assay method.

Rhamnose

Negative

Results

Cellobiose

Positive

Isolation and identification of isolated strain

Ribose

Positive

Lactic acid bacilli were selectively isolated from donkey milk

Galactose

Positive

sample. This LAB strain was identified as Lactobacillus

Trehalose

Positive

paracasei based on physiological, biochemical characteristics

Glucose

Positive

and sugar fermentation tests (Table 1).

The above tests confirmed the isolate as L. paracasei

Fig. 1a. Effect of temperature on bacteriocin production

Characteristics

Result

Colony Morphology

Creamy, round, convex, smooth

Colony Size

2-3 mm

pH

7.0

Temperature

Anaerobic, 37C

Morphology

Rods

Gram strain

Gram positive

Motility

Non-motile

Optimization assay for bacteriocin production

The selected pure culture of L. paracasei showed optimal
growth in MRS broth with an optimal bactericidal protein
production was observed at pH 6.0 and 1.5% NaCl when
cultures were incubated at 40C (Figs. 1a, 1b and 1c).

2500
2000
1500
1000
500
0
20C

30C

40C

50C

60C

Tem perature

Fig. 1b. Effect of pH on bacteriocin production

3000

Bacteriocin Prodiuction
(Au/ml)

Lactobacillus paracasei

Bacteriocin Production
(AU/ml)

Table 1a. Morphological and physiological characteristics of

2500
2000
1500
1000
500
0
4

pH

Table 1b. Biochemical characteristics

Fig. 1c. Effect of NaCl on bacteriocin production
Result

Catalase

Negative

Oxidase

Negative

Indole

Negative

Methyl red

Negative

Voges proskauer

Negative

Citrate Utilization

Negative

The above tests confirmed the genus as, Lactobacillus

3000

Bacteriocin Production
(AU/ml)

Biochemical Test

2500
2000
1500
1000
500
0
0.5

1.5

Sodium chloride (%)

2.5

Ashokkumar et al., 2011

Screening for antibacterial susceptibility pattern

Staphylococcus aureus and Escherichia coli.

The susceptibility of various pathogenic organisms to growth

inhibition by CFS of L. paracasei is shown in Table 2.

Fig. 2. Pathogenic bacterial inhibition by CFS

Table 2. Inhibition of organisms by bacteriocin produced by

L. paracasei
Clinical Isolates

Degree of Inhibition

Escherichia coli

+++

Klebsiella pneumoniae

+++

Pseudomonas aeruginosa

+++

Salmonella typhi

+++

Staphylococcus aureus

++

++ Optimal inhibition, +++ Maximum inhibition

Maximum inhibition denoted by +++ inferred that growth of the

test pathogenic bacteria was not observed even at dilution as low
as 10-2. While, optimal inhibition denoted by ++ inferred

Protein estimation

bactericidal inhibition being observed in the range of 10 -2 and

Protein estimation in the CFS and partially purified protein in

10-4 dilution. The highest inhibitory activity was demonstrated

the dialysate was performed by Lowrys method is illustrated in

against Salmonella typhi, Klebsiella pneumoniae, Pseudomonas

Fig. 3a. The optical density (O.D) at 600 nm for protein content

aeruginosa and Escherichia coli, while, the lowest inhibitory

in CFS was 0.66 while, for partially purified protein in the

activity of the cell-free supernatant was demonstrated against

dialysate obtained by ammonium sulphate precipitation and

Staphylococcus aureus (Fig. 2).

dialysis was 0.24.

Table 3. MIC of CFS against test pathogens

Protein estimation in fractionates obtained by IEC: Ion

exchange chromatography of dialysate resulted in collection of

20 purified protein fractionates.

isolates

Fig. 3a. Protein estimation in CFS (T1) and in dialysate (T2)

Concentration of Crude Bacteriocin (l)

Clinical

1024

512

256

128

64

32

16

MIC

512

256

256

128

512

Protein estimation by Lowrys methods supported the selection

of tubes containing maximum purified protein bacteriocin was
identified by using spot assay method. Primarily, the protein
estimation by Lowrys method demonstrated in Fig. 3b, three

(-): No growth of clinical isolate; (+): Growth of clinical isolate

major O.D. values at 600 nm corresponding to Tube No.: 5

MIC assay

(0.13), 8 (0.11) and 10 (0.09).

The MIC assay of CFS of L. paracasei was performed and is

Screening for bacteriocin in IEC fractionates: Spot assay was

shown in Table 3. The results indicated that MIC was 128 l

employed to qualitatively evaluate the fractionate containing the

against Salmonella typhi, 256 l against Pseudomonas

purified bacteriocin. Three fractionates i.e. Tube No.: 5, 8 and 10

aeruginosa

were selected for Spot assay method based on the Lowrys

and

Klebsiella

pneumonia,

512

against

Ashokkumar et al., 2011

protein estimation peak as illustrated in Graph 3b. The

et al., 2010). Moreover, Nutritional levels of donkey milk are

fractionate in Tube No. 5 contained pure bacteriocin based on

greater than those in milk from other sources (Salimei et al.,

the resulting Spot assay which showed maximum zone of

2004). Todorov and Dicks (2004) have claimed that bacteriocin

inhibition in this particular vial (Fig. 4).

production is strongly dependent on pH, temperature and

Fig. 3b. Protein estimation in tubes containing IEC fractionates

nutrient levels of the culture environment. In the present study

(T1 - T20) and B: Blank

optimal growth was observed in MRS broth and the optimal

production of bacteriocin isolated was observed at pH 6.0 and
1.5% NaCl when cultures were incubated at 40C. This is in
consensus with the studies conducted by (Karthikeyan and
Santosh, 2009, Ogunshe et al., 2007)
The isolated Lactobacillus paracasei was used for separation of
CFS containing extracellular crude bacteriocin. This CFS was
tested for antimicrobial susceptibility to a spectrum of
pathogenic

Gram-positive

and

Gram-negative

bacteria

commonly known to be associated with various clinical

manifestations by Spot assay method. The highest inhibitory
Fig. 4. Pathogenic bacterial inhibition by IEC fractionate in
Tube no: 5

activity was demonstrated against Salmonella typhi, Klebsiella

pneumoniae, Pseudomonas aeruginosa and Escherichia coli
while, the lowest activity seen against Staphylococcus aureus.
The inhibitory activity demonstrated by crude bacteriocin
against these organisms is a firm indicator of the presence of
active bacteriocin in the test supernatant. The inhibitory activity
of bacteriocins has also been reported against a number of other
bacteria (Ogunbanwo et al., 2003). Similar results have been
observed in experiments related to inhibitory effect of
bacteriocin produced by other Lactobacillus species (Tatsadjieu
et al., 2009).
The CFS extracted in the present study showed maximum
antimicrobial activity against Salmonella typhi which was also

Discussion
isolation,

well supported by the results obtained in the MIC assay with an

characterization of Lactobacillus paracasei from donkey milk

index of 128 l. The MIC assay conducted resulted in an index

sample and the activity of bacteriocin produced by selected

of 256 l against Klebsiella pneumoniae and Pseudomonas

strain. The potential of milk as a LAB substrate has been

aeruginosa and, 512 l against Escherichia coli and

demonstrated in an earlier study on fermented milk in Burkina

Staphylococcus aureus. In an earlier study conducted by Tzu-

Faso which reported isolation of 98 LAB strains out of 100

Hsing Lin, cell-free supernatants of L. paracasei sub sp.

strains cultured from milk (Aly et al., 2004). Various LAB

paracasei NTU101 showed maximum inhibitory effects on

species have been isolated and studied previously from cow and

pathogenic strains especially against Vibrio paraheamolyticus

goat milk samples. In an earlier study, Lactobacillus brevis, a

followed by a optimum inhibition against Klebsiella pneumoniae

bacteriocin-producing strain, has been isolated from goat milk

and Pseudomonas aeruginosa, the latter as observed in the

and was characterized as mesophillic hetero-fermentative

present study where 256 l of crude bacteriocin was effective

(Bettache and Mebrouk, 2004). Recently, donkey milk has been

against these organisms (Klaenhammer, 1983; Flythe et al.,

considered to be a potential alternative to cow milk for feeding

2004). The present study also included purification of

children, as the latter in some cases has been reported to cause

bacteriocin from the extracellular CFS. Lowrys method was

severe allergy in some children (Cavataio et al., 1996; Filomena

employed to estimate the protein content of the CFS, the

The

present

investigation

highlights

the

Ashokkumar et al., 2011

partially purified dialysate obtained through ammonium sulphate

Cavataio F, Iacono G (1996). Clinical and pH-metric

precipitation and the fractionates obtained from IEC. The graph

characteristics of gastro-oesophageal reflux secondary to

of the protein estimation of the fractions indicated 3 tubes

cows' milk protein allergy. Arch of Dis in Ch hood 75: 51-

containing presence of bacteriocin.

56.

These were selected for

qualitative screening of bacterial growth inhibition by Spot

Corsetti A, Gobetti M, Rossi J, Damiani P (1998). Antimould

assay method. Fractionate in Tube no: 5 demonstrated a clear

activity of Sourdough lactic acid bacteria- Identification of

zone of inhibition which confirmed the presence of bacteriocin

a mixture of organic acids produced by Lactobacillus

in this tube.

sanfranciso CB1. Appl Microbiol Biotechnol 50: 253-256.

The present study thus inferred that Lactobacillus paracasei

Devugst L, Vandamme E (1994). Bacteriocins of Lactic acid

isolated from donkey milk produced bacteriocin. The presence

bacteria: Microbiology, Genetics and Application. Blackie

of this protein was confirmed by the optimization assay

Academic and Professional Publishers. London, UK pp.

supportive for bacteriocin production and the activity was

91-142.

demonstrated by Spot assay and MIC assay. The study also

Elisabetta Tome, Vera L, Pereira, Carla I, Lopes, Paul A, Gibbs

followed isolation of pure bacteriocin fractionates using IEC and

Paula C, Teixeira (2008). Invitro tests of suitability of

confirmed its bactericidal activity qualitatively by Spot assay.

bacteriocin-producing lactic acid bacteria, as potential bio

The bacteriocin of Lactococcus lactis added in model cultured

preservation cultures in vacuum - packaged cold-smoked

cow milk, showed no spoilage upto 6 days against Listeria

salmon. Food Control 19: 535-543.

monocytogens (Benkerroum et al., 2002). Moreover, some of the

Filomena N, Pierangelo O, Florinda F, Raffaele C (2010).

other bacteriocins like Nisin have been approved by Food and

Isolation of Components with Antimicrobial Property from

Drug Administration as safe bio-preservative in pasteurized

the Donkey Milk: A Preliminary Study, The Open Food Sci

processed cheese (Rossland et al., 2005). These observations

J 4: 43-47.

make the bacteriocin produced by L. paracasei, a potential

Flythe MD, Russsell JB (2004). The effect of pH and a

substance to be used in bio-preservation, and also as a potent

bacteriocin (bovicin HC5) on Clostridium sporogenes

probiotic against human, animal, and avian pathogens. Further

MD1, a bacterium that has the ability to degrade amino

studies can be directed towards understanding the protein

acids in ensiled plant materials. FEMS Microbiol Ecol 47:

structure and genomics for greater production of bacteriocin

215-22.

from L. paracasei for making way for better application

Garvie E (1986). Genus Leuconostoc, In Sneath PHA, Mair NS,

avenues.

Sharpe ME, Holt JG editors. Bergeys Manual of

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