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Journal of Herbal Medicine and Toxicology 4 (2) 167-175 (2010)

ISSN : 0973-4643 Original Article

ENHANCEMENT OF ANTIMICROBIAL POTENTIAL OF

PHYLLANTHUS NIRURI BY FERMENTATION

Vaishnavi Venugopalan, Dinesh M.S.* and Geetha K.S.

Department of Biotechnology, PES Institute of Technology, 100 feet Ring Road, BSK III Stage, Bangalore

*Corresponding author - E-mail: dineshms@pes.edu

Received - 5th April, 2010; Revised - 4th May, 2010; Accepted - 28th May, 2010

ABSTRACT: The use of plants products for medicinal value is common in Indian

system of medicine. We can enhance the medicinal property of plants throughbioprocesses. In the present study, bioprocess such as fermentation was used to enhancethe antimicrobial potential of the crude herbal extract of Phyllanthus niruri.

Fermentation was carried out using the standard/commercial isolates of Lactobacillusacidophilus and by the isolates of the same bacterium from the herb surface separately.The fermented product obtained from both these procedures were compared for their

antimicrobial property against common human pathogens like Escherichia coli,Staphylococcus aureus, Salmonella typhi, Psuedomonas aeruginosa, Bacillus cereus,Bacillus subtilus and Klebsiella species. The variation of the antimicrobial property of

the fermented extracts along the fermentation time period was also studied. The resultsindicated that the antimicrobial potential of the fermented herb is more than that ofthe crude herbal extract. The antimicrobial property of the fermented herb increasesby about 80% -170% when compared to the crude herbal extract. Also, the fermented

product obtained using Lactobacillus isolates from the herbal surface is more potentagainst tested human pathogens when compared to the product obtained usingcommercial L.acidophilus isolates. The potency improves by 49% if Lactobacillus

species from the herbal surface are used for fermentation. The antimicrobialcapabilities of the fermented product increases along the fermentation period by about65%-95% irrespective of the source of lactic acid bacteria used. Another salient feature

of the study is that E.coli is the most sensitive while Klebsiella species is the leastsensitive to both the crude as well as the fermented extracts.

Key Words: Phyllanthus niruri, Lactobacillus isolates, Fermentation, Antimicrobial

activity

INTRODUCTION

Plants have always been a common source of

medicaments, either in the form of traditional

preparations or as pure active principles. Medicinal

plants are considerably useful and economically

essential. They contain active constituents that are

used in the treatment of many human diseases [1].

Many plant extracts have been developed and

proposed for use as antimicrobial substances. Plants

used in traditional medicine contain a vast array of

substances that can be used to treat chronic and

infectious diseases [2]. Plants have an almost limitless

ability to synthesize aromatic substances, most of

which are phenols or their oxygen-substituted

derivatives. Most are secondary metabolites, of which

at least 12,000 have been isolated, a number estimated

to be less than 10% of the total [3]. In many cases,

these substances serve as plant defense mechanisms

against predation by microorganisms, insects, and

herbivores. Some, such as terpenoids, give plants their

odors; others (quinones and tannins) are responsible

for plant pigment. Many compounds are also

responsible for plant flavor while some of the herbs

and spices used by humans to season food yield useful

medicinal compounds [4]. Phyllanthus niruri is a

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168

herb belonging to Euphorbiaceae family. It is known

for a wide variety of phytochemicals and

pharmacological properties. It is an ingredient of

almost 175 ayurvedic formulations. Fruits are

commonly used in the treatment of hemorrhages,

diarrheas, dysentery, jaundice, cough and anaemia. It

is also used for the preparation of various health care

and personal products like chavanprash, hair oil, dye,

face cream, tooth powder etc [5]. The active

phytochemicals, flavonoids, alkaloids, terpenoids,

lignans, polyphenols, tannins, coumarins and saponins,

have been identified from various parts of P. niruri.

Extracts of this herb have been proven to have

therapeutic effects in many clinical studies [6].

Fermentation of herbs produces beverages which are

considered to be healthy by people who consume it.

They contain high nutritional content and bioactive

compounds formed from the raw material and the

fermentation reactions [7]. Lactic acid fermentations

is regarded the most popular used for fermenting of

herbs. Lactic acid bacteria (LAB) produces organic

acids, diacetyl, hydrogen peroxide and bacteriocins

that improve the shelf life of the fermented product

by controlling spoilage organisms and potential

pathogens. LAB is also known to possess

antimicrobial properties thereby improving human

health [8, 9]. In the present study, a crude extract of

P.niruri was tested against common human pathogens

using a standard antibiotic as positive control and

sterilized distilled water as a negative control. Further

to this, the herb was separately fermented using

Lactobacillus species isolated from two different

sources – standard or commercially available

Lactobacillus acidophilus and the Lactobacillus

sp. isolated from the herbal surface. The antimicrobial

activities of the fermented products obtained from

both these bacterial strains were then compared and

evaluated. The activities along the fermentation period

(25 days) were also studied comparatively.

MATERIAL AND METHODS

Plant material: The fresh herbs were collected from

the aromatic crop section, Division of Horticulture,

UAS (University of Agriculture Sciences), GKVK,

Bangalore, India, for the present study. Fresh herbs

that are not infected by pests and disease damage

were harvested. The parts of the herbs like leaves

and fruits were detached and washed thoroughly with

clean water.

Preparation of the crude extract: 20g of clean and

fresh herbs were crushed using a pestle and mortar

and added to 100 ml sterile water to obtain an aqueous

extract of the herb (200mg/ml). The extraction was

done at room temperature (24 °C). Muslin cloth was

then used to filter the plant residues and the filtrate

thus obtained was further purified by filtration through

Whatman No 1 filter paper [10, 11].This crude extract

was tested against common human pathogens for

antimicrobial properties.

Culturing of Lactobacillus isolates for fermentation

of the herb: Standard or commercial Lactobacillus

acidophilus isolates were obtained as pure cultures

from the Department of Microbiology, G.K.V.K,

Bangalore, and stored in semi solid medium at 4° C

for further utilization in the study. Before use, there

were thawed and maintained in Mann Rogosa

Sharpe’s media [12]. In order to isolate Lactobacillus

species from the herbal surfaces, clean and washed

leaves of the herb were placed on Mann Rogosa

Sharpe’s agar (15 ml) in petri plates under sterile

procedures [13]. MRS agar is used for isolation of

lactic acid bacteria species since the medium restricts

growth of other species [12]. The plates were

incubated at 37°C for 2 days. On observing the petri

plates, uniform colonies of bacteria were seen to be

grown. These colonies were similar in morphology

and other physical characteristics as that of standard

Lactobacillus acidophilus colonies maintained. The

colonies isolated from the herbal surfaces were

identified and characterized using standard methods

such as gram staining, test for catalase, gelatin

hydrolyses activities and ability to assimilate a range

of carbon sources [13, 14]. Hence, the standard and

the herbal isolates of Lactobacillus species were

separately used to ferment the herb extract of

Phyllanthus niruri to know their efficacies. The

characteristics of the two lactic acid bacterial isolates

are outlined in table 1. The isolates used in the study

are shown in figure 1.

Lactic acid fermentation of the herb [15-17]:

The following sequential steps were followed.

(i) Preparation of P.niruri based fermentation media:

The pest and disease free leaves of the herb were

harvested after eliminating its surface microbial load

using running water. 20% of the medicinal herb was

considered for fermentation. 20g of leaves were

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Venugopalan et al.

Table 1: Characteristics of the Lactic acid bacteria (LAB) isolates collected from two different

sources

Characters tics studies Source of LAB Isolates

Standard / Commercially

available LAB

LAB isolated from herbal

surface of Phyllanthus niruri

Cell Shape and Arrangement Rods Rods occurring in short chain

Gram Reaction Positive Positive

Catalase Reaction Negative Negative

Gelatin Hydrolysis Positive Positive

Growth on MRS broth after 24 hrs of incubation (OD at 600nm) 1.25 1.23

Growth on MRS broth after 48 hrs of incubation (OD at 600nm) 1.71 1.72

Assimilation of Glucose Good growth Good growth

Assimilation of Sucrose Medium growth Medium growth

Assimilation of Galactose Medium growth Medium growth

Assimilation of Lactose Medium growth Medium growth

Organism Lactobacillus

acidophilus

Lactobacillus Sp.

The above table shows the different characteristics studied for the two sources of Lactic acid bacteria (LAB) isolated in the

present research work – one commercially available and the other from the surfaces of the herb Phyllanthus niruri.

Table 2: Antimicrobial Activities of crude herbal extract of P.niruri and fermented products

against common human pathogens

Bacterial

Pathogens

Zone of

Inhibition

(mm) for

NC

Zone of

Inhibition

(mm) for

CEx

Zone of Inhibition (nm) at different fermentation times Zone of

Inhibition

(mm) for

PC

Day 5 Day 10 Day 15 Day 20 Day 25

A B A B A B A B A B

E. Coli 0.0 6.0 6.5 8.0 8.5 9.5 9.0 11.5 10.5 14.0 10.5 14.0 2.5

S. aureus 0.0 5.5 6.0 7.5 7.5 8.5 8.0 9.5 9.0 12.0 9.1 12.0 20.5

S. thphi 0.0 4.0 4.0 4.5 5.0 5.5 6.0 7.0 6.5 9.0 6.5 9.2 2.5

P.aeruginosa 0.0 2.0 2.5 3.0 3.5 4.0 3.5 6.5 4.0 8.5 4.1 8.5 1.5

B.cereus 0.0 1.5 2.0 2.5 2.0 3.5 3.0 5.0 3.5 6.0 3.5 6.1 14.0

B.subtilus 0.0 1.5 1.5 2.0 2.0 2.5 2.5 3.5 3.0 4.5 3.0 4.5 15.1

Klebsiella sp. 0.0 0.5 1.0 1.5 1.5 2.0 2.0 2.5 2.5 3.5 2.6 3.5 6.1

NC: Negative control (distilled water); CEx: Crude Extract of the herb Phyllanthus niruri; PC: Positive control

(Ampicillin taken 10 µg/disc); A: Fermented products obtained using Standard/commercially available LAB

isolates ; B: Fermented products obtained using LAB isolated from the herbal surfaces of Phyllanthus niruri. The

above table shows the antimicrobial activities of the controls and the samples against common human pathogens.

Fermented samples are tested every 5 days for a period of 25 days of the fermentation period. The tests were

performed in triplicates and the average value has been entered in the above table.

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Venugopalan et al.

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crushed with pestle and mortar and added to 100ml

of sterile distilled water. The extract was filtered using

muslin cloth and Whatman No 1 filter paper to remove

the plant debris present. 400 ml of this herbal extract

was made and stored in conical flasks (100 ml each),

to be used for preparing starter cultures and for lactic

acid fermentations. The Lactobacillus species

(standard isolates and herbal surface isolates)

collected and maintained previously were used

separately to ferment the herbal extract prepared.

Before subjecting the herbal extract to fermentation,

each of the Lactobacillus isolates were multiplied

and made to adapt to herbal environment by preparing

respective starter cultures. (ii) Preparation of starter

culture of Lactobacilli strains: To prepare a starter

culture, a test tube with 5ml of sterilized MRS broth

was added with one loop inoculums of the lactic acid

bacterial culture intended to be multiplied and kept

overnight at 25 °C for growth. The culture grown in

the test tube was then added to 100ml of herbal extract

in a conical flask. The flask was kept at 37 °C in an

incubator with shaking facility of 100rpm for 24 hrs.

Dilution was done using MRS broth to obtain a size

of 106 cfu/ml. Adjusted cultures was tested for right

size by plate count method on MRS agar. 5ml (v/v)

of the adjusted starter culture was used for further

study. Respective starter cultures were used for

respective fermentations keeping the number of lactic

acid bacterial cells added to herbal extract constant.

(iii) Setting up the fermentation process: 100 ml of

the herbal extract in a conical flask was fermented

using standard Lactobacillus acidophilus starter

culture and 100 ml of herbal extract in another conical

flask was fermented using starter culture of

Lactobacillus isolates from the herbal surface. About

2g of dextrose was added to enhance the fermentation

process. Fermentation process was carried out at room

temperature using a shaker incubator (100rpm). The

fermented products from each of these were

compared for antimicrobial activity at every 5 days

interval for a period of 25 days. A flow chart of the

fermentation procedure is shown in fig 1.

Collection and culturing of human pathogens: The

microorganisms to be tested against the crude herbal

extract and the fermented products of P.niruri were

obtained from the Department of Microbiology,

G.K.V.K., Bangalore, as pure cultures maintained in

nutrient agar medium at 4°C for timely use. The

human pathogens collected were Escherichia coli,

Staphylococcus aureus, Salmonella typhi,

Psuedomonas aeruginosa, Bacillus cereus,

Bacillus subtilis and Klebsiella species. Suspension

cultures of the pathogens were made prior to testing

the antimicrobial potential of the fermented products.

Nutrient broth was used for the preparation of bacterial

culture suspension [18]. Using a sterile loop, 3-5

colonies of the bacterial cells were transferred from

the stock culture plate into a 10ml of nutrient broth

taken in test. The test-tube was incubated at 37°C

for 24 hours. Using sterile nutrient broth, a population

of 106 cfu/ml was obtained for each pathogen.

Appropriate dilution was determined after spread

plating the adjusted culture on Plate Count Agar

(PCA). 100 µl of 24 hr incubated test organism was

used for further studies [19]. All the growth media

used in the experiment was purchased from Himedia

Laboratories Pvt. Ltd., Mumbai.

Screening the crude herbal extract and fermented

products for antimicrobial activity: To test the

antimicrobial potential of fermented products, modified

agar diffusion method (filter paper disc method) was

employed [20, 21]. On solid nutrient agar medium (15

ml taken in petri plate), the pathogenic culture

suspension was swabbed uniformly. A sterile filter

paper impregnated with 0.1ml of the sample to be

tested for antimicrobial activity was placed on the

agar surface and kept for incubation at 37°C for 24

hrs. After the stipulated time, presence of a clear zone

of inhibition around the filter paper disc indicated the

sensitivity of the pathogen to the sample tested [22,

23]. Sterile distilled water was taken as negative

control while ampicillin (10µg/ml) was taken as the

positive control. The zone of inhibition was calculated

by subtracting the diameter of the zone formed due

to application of the sample on the filter paper disc

from the diameter of filter paper disc used for testing

sensitivity of the pathogens to the sample [21]. The

experiment was done in triplicates and the zones of

inhibition seen were reported in mm.

RESULTS AND DISCUSSION

(i) Isolation and collection of Lactobacillus species:

The characteristics of Lactobacillus species collected

from standard sources and the ones isolated from

herbal surfaces were studied by growth on MRS agar.

These include features like colony morphology, gram

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Venugopalan et al.

staining, catalase activity, gelatin hydrolysis activity

and capability to assimilate various sugars. The results

of the same are outlined in table 1. From the table it

can be seen that species isolated from the herbal

surfaces bears same features as that of the

commercially available L.acidophilus species. Hence

it was reported that the species isolated from the herb

on MRS agar is Lactobacillus species. Literature

shows that studies of isolating lactic acid bacteria from

different herbs gave similar results as the present

study. J. Orvin Mundt and James L. Hammer isolated

Lactobacillus casei, L.leichmanii, L.platarum from

chickpeas and cowpeas in 1968 [24]. Cereals and

millets contain lactic acid bacteria species which were

successfully isolated on MRS media [25, 26].

(ii) Screening and evaluation of antimicrobial activity

of crude extract P.niruri: The antimicrobial assay

done by filter paper disc procedure showed that the

crude herbal extract of Phyllanthus niruri exhibits

antibacterial activity against all the tested pathogens.

During the assay, sterile distilled water was used as

negative control while ampicillin was used as positive

control. The zone of inhibition in mm for the crude

extract is shown in table 2. From the table it can be

seen that the zones of inhibition formed by the crude

extract and standard antibiotic (ampicillin) are

comparable. Results showed that out of the 7

microorganisms tested, E.coli, S.typhi and

P.aeruginosa were most sensitive to the crude extract

when compared to the standard antibiotic. The herb

Phyllanthus niruri is a rich source of phytochemicals,

including many which have been found only in the

Phyllanthus genus [27]. Many of the constituents are

attributed to biologically active lignans, glycosides,

flavonoids, alkaloids, ellagitannins, and

phenylpropanoids found in the leaf, stem, and root of

the plant. Common lipids, sterols, and flavonols also

occur in the plant. The main plant chemicals in it

include alkaloids, astragalin, brevifolin, carboxylic

acids, corilagin, cymene, ellagic acid, ellagitannins,

gallocatechins, geraniin, hypophyllanthin, lignans,

lintetralins, lupeols, methyl salicylate, niranthin,

nirtetralin, niruretin, nirurin, nirurine, niruriside,

norsecurinines, phyllanthin, phyllanthine, phyllanthenol,

phyllochrysine, phyltetralin, repandusinic acids,

quercetin, quercetol, quercitrin, rutin, saponins,

triacontanal, and tricontanol [28].

(iii) Enhancement of antimicrobial activity: During

fermentation of P.niruri, the antimicrobial potency

was seen to be enhanced when the herbal extract

was fermented using lactic acid bacteria. This was

evident through comparing the zones of inhibition (mm)

formed against the tested pathogens by crude extract

and the fermented products (Table 2). Fermentation

was carried out separately by using standard

L.acidophilus isolates and Lactobacillus species

isolated from the herbal surfaces. Fermented products

of both the fermentation procedures gave

antimicrobial potential better than that of the crude

unfermented extract. In comparison to the crude

extract, fermentation of the herb increased the

antimicrobial activity by 80% when commercial

L.acidophilus were used and by 170% when herbal

isolates of Lactobacillus species were used. Many

phytochemicals important for antimicrobial activity in

a herb are less freely available and mainly exist bound

to the cell surface [30, 31]. Fermentation process

causes release of microbial enzymes which in turn

produce more freely available forms of plant

chemicals like flavonoids, alkaloids and

phenylpropanoids [32]. The increase in the levels of

free (non-bound) plant chemicals may be responsible

for the improvement in antimicrobial activities. Also,

the fermented products formed by the Lactobacillus

species from the herb surfaces gave better zones of

inhibition than the products formed by standard

L.acidophilus isolates. The antimicrobial property

was enhanced by about 49% when herbal isolates of

lactic acid bacteria was used for fermentation. This

could be because the Lactobacillus species isolated

from Phyllanthus niruri are native to the herb and

hence more adapted to the herb than the commercially

available L.acidophilus. Due to this, the potential to

ferment the herbal extract is better by former than

the latter thereby releasing more active fermentation

products that are better potent against the human

pathogens. The fermented products (irrespective of

the source of lactic acid bacteria) have a potential to

be commercialized since they showed better activities

against E.coli, S.typhi and P.aeruginosa than the

standard antibiotic that was used as positive control.

A very important result through the present study is

the increase in antimicrobial activity along the

fermentation period. Assaying was done every 5 days

to a period of 25 days, during which increase in zones

of inhibition can be clearly seen from table 2. This is

regardless of the source of lactic acid bacteria used.

Journal of Herbal Medicine & Toxicology

174

As the fermentation time progressed, antimicrobial

activity was increased by about 65% when

commercial L.acidophilus were used and by 95%

when herbal isolates of Lactobacillus species were

used. The activity, estimated in terms of the zone of

inhibition, reached a peak on the 20th day of

fermentation and remained steady thereafter. Since

all the available raw materials get converted to

fermented products by the 20th day of fermentation

process, the zones of inhibition formed by the samples

from then on remains constant [31]. A graphical

representation has been shown in figures 3, 4 and 5.

Among the organisms tested, the sensitivity of the

pathogens varies as follows:

E.coli>S.aureus>S.typhi>P.aeruginosa>B.cereus>B.subtilis>Klebsiella

species. The outer membrane of bacteria and its

structure are responsible for differences of sensitivity

that may occur between different bacteria [33, 34].

CONCLUSION

The results of the present study show that

fermentation of the herb Phyllanthus niruri evidently

enhanced the antimicrobial properties of the herb.

Also, using the Lactobacillus species growing on the

herbal surface if used for the fermentation process

gives better results for antimicrobial property. The

fermented P.niruri obtained using isolates of

Lactobacilli from the leaf surface have high potential

of being developed as a neutraceutical to curb

infectious diseases caused by the pathogens used in

the study.

ACKNOWLEDGMENT

The authors thank the Principal, Dr. K.N.

Balasubramanium, and the Management of PES

Institute of Technology, Bangalore India, supporting

the study through PESIT Internal project. We also

thank Dr. V. Krishnamurthy, Head of the Department,

Biotechnology, for providing useful suggestions to

improve our work. Lastly we thank the Department

of Microbiology and Horticulture, G.K.V.K., Bangalore

for providing us the materials required for the study.

REFERENCES

[1]. Stary, F., Hans, S.: The National guides to medical

herbs and plants. Tiger Books. Int. Plc. UK. (1998).

[2]. Rustomjee, N.K., Nanabhai, N.K.: Materia Medica of

India and their therapeutics. Komal Prakashan, BRPC

(India) Ltd. New Delhi. (1999).

[3]. Geissman, T. A.: Flavonoid compounds, tannins,

lignins and related compounds. In: Pyrrole pigments,

isoprenoid compounds and phenolic plant

constituents (M. Florkin, and E.H. Stotz ed.)., vol. 9.

Elsevier, New York, N.Y., pp 265-70 (1963).

[4]. Wild, R.: The complete book of natural and medicinal

cures. Rodale Press, Inc., Emmaus, Pa. (1994).

[5]. Rastogi, R.P. and B.N. Mehrotra: Compendium of

Indian medicinal plants, Vol. II. Central Drug Research

Institute, Lucknow and publications and Information

Directorate, New Delhi (1991)

[6]. Bagalkotkar, G., Sagineedu, S.R., Saad, M.S., Stanslas,

J.: J Pharm Pharmacol., 58(12):1559-70 (2006).

[7]. Battcock, M. and S. Azam-Ali: FAO Agric. Services

Bull., 14:134 (1998).

[8]. Adams, M.R., M.O. Moss: Food Microbiology. 2nd

edn. Royal Society of Chemistry, University of Surrey,

Guildford, UK (2003).

[9]. Oyeta, V.O., F.C. Adetuyi and F.A. Akinyosoye: Afr.

J. Biotechnol., 2(11): 448-452 (2003)

[10]. Sumathi, S. and A. Parvathi, A. : J Med Plants Res,

4(4): 316-332 (2010).

[11]. Sule, I.O. and T.O. Agbabiaka: Ethnobotanical

Leaflets, 12: 1035-42 (2008).

[12]. Rogosa, M., Sharpe, M.E.: J. Appl. Bacteriol. 22:329-

340 (1959).

[13]. Nilsson, G., Nilsson, P.E.: Arch. Mikrobiol., 24:412-

422 (1956).

[14]. Kandler, O.: Taxonomie und technologische

Bedeutung der Gattung Lactobacillus Beijerinck. In:

Arbeitsmethoden und actuelle Ergebnisse der

technischen Mikrobiologie. 3rd Symposium der

Gesellschaft, Berlin, 138-164 (1996).

[15]. Savadoga, A., Ouattara, C.A.T., Bassole, H.N.,

Traore, A.S.: Pak. J. Nutr., 3(3): 174-179 (2004).

[16]. Shelef, L.A.:. J. Food Prot., 57(5): 445-450 (1994).

[17]. Kantachote, D., Charemjiratrakul, W., Umsakul, K. :.

J. of Biological Sci., 24(1): 1-9 (2008).

[18]. Swanson, K.M.J., Busta, F.F., Peterson, E.H.,

Johnson, M.G.: Colony count methods. In:

Compendium of Methods for the Microbiological

Examination of Foods (C. Vanderzant and

Splittstoesser, D.F. eds.). 3rd ed. American Public

Health Association, DC, pp. 75-95 (1992).

[19]. Hugo W.B. and Russell A.D: Pharmaceutical

Microbiology. 5th Ed. Blackwell Scientific

Publications Oxford, London, 258-297 (1992).

[20]. Rios, J.L.,Recto, M.C., Villar, A.: J. Ethnopharmacol.,

23: 127-149 (1998).

[21]. Piddock, L.J.V:. J. Appl. Bacteriol., 68: 307-318 (1990).

[22]. Olowosulu, A.K. and Ibrahim, Y.K.E.: West African

J. boil. Sc., 3(5): 21-26. (2006).

175

[23]. Akinjobi, C.O. Anyanuwu, B.N., Onyeze, G. O. C.

and Ubekwe, V.I.:. Journal of Applied Science, 7 (3):

4328 – 4338 (2004).

[24]. Orvin Mundt, J., James A.D and L. Hammer:. J.

Applied microbiology, 16 (9): 1326-1330 (1968).

[25]. Sulochana M.B., Sharda, B., Dayanand., A.:

Detection and development of Lactobacilli in cereals

and their features. In: Biotechnology of Microbes

and Sustainable Utilization Edited (Prof. R.C. Rajak).

Scientific Publishers, Jodhpur, pp 239-243 (2002).

[26]. Sulochana M. B. and Dayanand A.: Journal of

Microbial World 5(1): 39 – 43 (2003).

[27]. Ekwenye, U.N., Njoku, N.U.: Antimicrobial effect of

Phyllanthus niruri (Chanca Piedra) on human

enteropathogens. Int. J. Mol. Med. Adv. Sci., 2(2):

184-189 (2006).

[28]. Chauhan, J.S. Sultan,M: Srivastava,S.K.:. J Indian

Chem. Soc., 68(80): 479-480 (1991).

[29]. Kuwakia S., Ohhiraa, I.,Takahatab M., Hirotaa, A.,

Murataa, Y., Tada, M.: J Biosci. Bioengin., 98(3): 187-

192 (2004).

[30]. Soo Joo, S., Joon Won, T., Nam, S.Y., Kim, Y-B., Chu

Lee, Y., Park, S-Y., Park, Kwang, H.Y., Hwang, W., Ik

Lee, Do:. J. Phytother. Res., 23(7): 913-919 (2009).

[31]. Kuwaki, S., Ohhira, I., Takahata, M., Murata, Y., Tada,

M. : J. Biosci. Bioengin., 94(5): 401-405 (2002).

[32]. Messens, W., Vuyst, L. De. :. Int. J. Food Microbiol.,

72(1-2): 32-43 (2002).

[33]. Gao, Y., Belkum, M.J.V., Stiles, M.: Applied Environ

Microbiol., 65(10): 4239-4333 (1999).

[34]. Pelczar, M.J.,. Chan, E.C.S. Jr., Kreig, N.R.:

Microbiology. Int. Edn. McGraw-Hill (1986).