O R I G I N A L A R T I C L E Technological properties and probiotic potential of Enterococcus...

ORIGINAL ARTICLE

Technological properties and probiotic potential ofEnterococcus faecium strains isolated from cow milkK. Banwo1,2, A. Sanni1 and H. Tan2

1 Department of Microbiology, University of Ibadan, P.M.B 1 Ibadan, Oyo State, Nigeria

2 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

Keywords

enterocin A, Enterococcus faecium, milk,

Nono, safety.

Correspondence

Kolawole Banwo, Department of

Microbiology, University of Ibadan, P.M.B 1

Ibadan, Oyo State, Nigeria.

E-mail: [email protected]

2012/1083: received 14 June 2012, revised

29 August 2012 and accepted 29 September

2012

doi:10.1111/jam.12031

Abstract

Aim: To identify enterococci from the fermentation of milk for the

production of nono, an African fermented dairy product, to determine the

technological properties for suitability as starter cultures and safety as

probiotics.

Methods and Results: Enterococcus faecium CM4 and Enterococcus faecium

2CM1 were isolated from raw cow’s milk. The strains were phenotypically and

genotypically identified. Technological properties, safety investigations, in vitro

adherence properties and antimicrobial characteristics were carried out. Strong

acidification and tolerance to bile salts were recorded. The strains were bile

salts hydrolytic positive and no haemolysis. There was no resistance to

clinically relevant antibiotics. The strains exhibited adherence to human

collagen type IV, human fibrinogen and fibronectin. The bacteriocins were

active against Bacillus cereus DSM 2301, Bacillus subtilis ATCC 6633,

Micrococcus luteus and Listeria monocytogenes. Bacteriocins were stable at pH 4

–9 and on treatment with lipase, catalase, a-amylase and pepsin, while their

activity was lost on treatment with other proteases. The bacteriocins produced

were heat stable at 100°C for 10 min. The bacteriocin produced by the strains

was identified as enterocin A.

Conclusions: The E. faecium strains in this study exhibited probiotic activity,

and the safety investigations indicate their suitability as good candidates for a

starter culture fermentation process.

Significance and Impact of the Study: The use of bacteriocin-producing

E. faecium strains as starter cultures in fermented foods is beneficial but,

however, their safety investigations as probiotics must be greatly emphasized.

Introduction

Enterococcus species are Gram-positive bacteria, faculta-

tive anaerobic cocci that occur singly, in pairs or in

chains (Giraffa 2003). They are commensal micro-organ-

isms that colonize in the gastrointestinal tract of humans

and animals and are also found in several different food

sources, such as meats, milk and cheese. These bacteria

are able to survive extreme environments, such as 6�5%NaCl, pH of 9�6, high heat as well as being able to grow

and survive under other harsh environmental conditions,

like those found in various soils, surface water, raw plants

and animal products (Giraffa 2003; Johnston and Jaykus

2004).

Enterococci are utilized in many diverse roles in a multi-

tude of different processes. For instance, they serve as an

important contributor in the ripening and flavour

enhancement of several types of food such as cheeses and

sausages (Giraffa 2003). In dairy products, their major role

is in the development of organoleptic characteristics during

the development and maturation process (Giraffa 2003;

Cocolin et al. 2007). These bacteria have also been utilized

as probiotics to improve the microbial balance of the intes-

tine and to treat gastroenteritis in humans and animals

Journal of Applied Microbiology 114, 229--241 © 2012 The Society for Applied Microbiology 229

Journal of Applied Microbiology ISSN 1364-5072

(Kayser 2003). Furthermore, enterococci harbour some

useful biotechnological and functional properties, such as

production of antimicrobials with antilisterial activity

(Foulquie-Moreno et al. 2003; Cocolin et al. 2007).

The antimicrobial properties of the strains have been

ascribed to the production of bacteriocins (Todorov and

Dicks 2006), which are defined as small proteins or pep-

tides with bactericidal or bacteriostatic activity against

genetically closely related species (Klaenhammer 1993).

There have been published reports on bacteriocin-pro-

ducing enterococci, mainly among the strains of Entero-

coccus faecium associated with food ecosystems and dairy

products (Cocolin et al. 2007).

There have been numerous reports on bacteriocin-pro-

ducing bacteria, primarily among strains of lactic acid bac-

teria (LAB) associated with food systems (Park et al.

2003), but there is little information on Enterococcus spe-

cies from African fermented food sources (Yousif et al.

2005) especially traditional fermented dairy products

(Olasupo et al. 1999; Yousif et al. 2005). Nono is fermented

cow milk taken with fura which is made from ground

millet. Hence, the name Fura de nono, African fermented

milk curd. This is consumed mainly by the northern region

of Nigeria and sub-Saharan Africa. The milk is either

consumed immediately after milking from the cow or left

to the next day. The method employed by the indigenous

people in the preservation of the milk is usually back slop-

ping (Eka and Ohaba 1977). Hence, the importance in the

development of starter culture fermentation process to

ensure consistency in the product and safety.

Bacteriocin production by LAB is believed to confer an

ecological advantage over competitors present in the

same ecosystem, as Enterococcus species form part of the

autochthonous flora of the intestinal tract of humans and

animals (Fuller 1989; Javed et al. 2011) and have also

been isolated from fermenting plant and dairy products

(Villani and Coppola 1994), they show potential for

application as probiotics. Their antibiotic susceptibility

profile also ensures their safety as probiotics especially

the absence of antibiotics and multidrug resistance genes

(Yousif et al. 2005). Enterococci from food sources have

relatively low virulence, and there are some beneficial

activities of some strains; they are also considered as nos-

ocomial pathogens, which cause bacteraemia, endocarditis

and other infections (Ben-Omar et al. 2004).

Production of bacteriocins by enterococci maybe an

advantage in the use as a probiotic because the bacterioc-

ins add up to antimicrobial effect of other substances

such as hydrogen peroxide and organic acids and con-

tribute to the competition in the gastrointestinal tract

(Strompfova and Laukova 2007). LAB strains that possess

certain functional properties such as tolerance to bile

salts, acidification, absence of antibiotic resistance genes,

in vitro adhesion capabilities and also production of bac-

teriocins render them good candidates for starter culture

fermentation process and safety as probiotics (Giraffa

2003; Yousif et al. 2005; Strompfova and Laukova 2007;

Javed et al. 2011).

In our study, we evaluated the probiotic potentials and

the bacteriocin production of two newly isolated Ent. fae-

cium strains from fermenting raw cow’s milk in Nigeria.

The aim of the study was to investigate the probiotic

properties, functionality and safety of the Ent. faecium

strains, to employ them as starter cultures for the

production of nono African fermented dairy product.

Materials and methods

Identification of bacteriocin-producing strains

Seven isolates from raw milk were characterized pheno-

typically as Enterococcus species. The strains were grown

in De Man, Rogosa, Sharpe agar (Oxoid, Basingstoke,

UK) at 37°C for 24–48 h. They were presumptively iden-

tified by the following tests: observation of colonial char-

acteristics and cell morphology, Gram staining, catalase,

growth at 15 and 45°C, growth in the presence of 6�5%NaCl and at pH 9�6 and fermentation of wide range of

sugars. Genetic identification to species level was per-

formed by 16S rRNA sequencing, as described by Brosius

et al. (1978) and Kostinek et al. (2005).

Acidification

The Enterococcus faecium strains were inoculated (1% of

an overnight culture) into MRS broth adjusted to pH

6�75 before autoclaving (pH 6�54 after autoclaving) and

grown aerobically at 30°C. Acid production was deter-

mined by measuring the pH of the culture after 6, 24

and 48 h. The MRS broth medium for all acid produc-

tion tests was prepared from a single batch that was pH

adjusted and then dispensed into tubes of 10 ml each

before autoclaving (Kostinek et al. 2005).

Resistance to bile salts

The ability of the strains to grow in the presence of bile

was determined according to the method of Walker and

Gilliland (1993) as modified by Vinderola and Reinhei-

mer (2003). Each strain was inoculated (2% v/v) into

MRS broth with 0�3, 0�5 and 1% (w/v) of bile (Sigma-

Aldrich, Saint Louis, MO, USA). Cultures were incubated

at 37°C for 24 h; optical density (OD) of the inoculated

tube was measured at OD560 nm wavelength and

compared with a control culture (without bile salts). This

was expressed as the percentage of growth at OD560 nm in

230 Journal of Applied Microbiology 114, 229--241 © 2012 The Society for Applied Microbiology

Enterococcus faecium strains from milk K. Banwo et al.

the presence of bile salts compared with the control. Each

determination was carried out in triplicates.

Bile salts hydrolytic activity

Bile salt hydrolytic (BSH) plates were prepared by adding

0�5% (w/v) sodium glycodeoxycholate (GCDA) and 0�5%(w/v) taurodeoxycholate (TDCA) to MRS agar. The

organisms were streaked on the plates and incubated at

37°C for 24–48 h, and the activities were determined by

the precipitation of deconjugated bile salts around the col-

onies, which was recorded as positive (Minelli et al. 2004).

Haemolysis and production of gelatinase

The strains were cultured in MRS broth at 37°C for 12

–18 h and then transferred onto blood agar (Difco, Michi-

gan, USA) plates supplemented with 5% defibrinated

whole sheep blood as described by Yoon et al. (2008).

After 24–48 h, the haemolytic reaction was recorded by the

observation of a partial hydrolysis of red blood cells and

greening zone (a-haemolysis), clear zone around bacterial

growth (b-haemolysis) and no reaction (c-haemolysis).

To determine the presence of gelatinase activity, the

plate assay method on Todd–Hewitt agar containing 30 g

of gelatin l�1 was carried out as described by Ben-Omar

et al. (2004).

Antibiotic susceptibility testing of the Enterococcus

faecium strains

The antibiotic strips for the determination of susceptibil-

ity profile were used according to the manufacturer’s

instructions. The susceptibility was determined towards

ten antibiotics, namely amoxycillin (25 lg), ofloxacin

(5 lg), streptomycin (10 lg), chloramphenicol (30 lg),cefriazone (30 lg), gentamicin (10 lg), pefloxacin (5 lg),cotrimaxazole (25 lg), ciprofloxacin (10 lg) and erythro-

mycin (5 lg). Tetracycline and vancomycin were tested

separately in a dilution of 30 lg ml�1 and 30 lg ml�1 in

sterile distilled water, respectively.

This was determined semiquantitatively using the agar

overlay diffusion methods of National Committee for Clin-

ical Laboratory Standards (NCCLS 1990). A bacterial sus-

pension was made by picking colonies from MRS agar

plates using a sterile loop and making a suspension in ster-

ile MRS broth and left for 18–24 h. With the use of a sterile

swab, the suspension was applied on the surface of Mueller

–Hinton agar in sterile Petri dishes, and the strips were

placed on the surface using a sterile scalpel. For tetracycline

and vancomycin, a 3-mm sterile cork borer was used after

which 100 ll of the antibiotic solution was dispensed into

the holes. The plates were then incubated at 30°C for 48 h

after which the readings were taken (Mueller and Hinton

1941; Hummel et al. 2007; Mathara et al. 2008).

In vitro adherence assay

This assay was carried out according to the study by Schil-

linger et al. (2005) with slight modification. Commercial

precoated 96-well human fibronectin (Fn) and human col-

lagen type IV cell culture plates were obtained from

Becton-Dickinson (San Jose, CA, USA) and human plasma

fibrinogen (Fb) (Sigma-Aldrich). Human plasma fibrino-

gen was dissolved in 0�1 mol l�1 phosphate-buffered saline

(PBS, pH 7�4) at a concentration of 50 lg ml�1 and used

to coat 96-well cell culture microtiter plates from Corning

(Lowell, MA, USA). The strains were grown in Dulbecco’s

modified Eagle’s medium (DMEM; Invitrogen, Beijing

China) supplemented with 2% (v/v) foetal bovine serum

(FBS; MDgenics, USA) for 24 h at 37°C. Cells were har-

vested by centrifugation at 10 000 g for 5 min and the pel-

lets resuspended in DMEM and diluted in 10�4 dilution

factor in Tris/borate/EDTA (TBE) buffer pH 8�0, and Syto

62 red (Molecular Probes, Eugene, OR, USA) was added as

the fluorescent dye for detection in the flow cytometer.

The cell count was determined using a BD FACS cell sorter

(BD Biosciences, San Jose, CA, USA). A 1-ml aliquot of the

bacterial suspension was adjusted to 1 9 108 cfu ml�1.

The wells were filled with 200 ll of extracellular matrices

(ECM) protein (Fb, Fn and collagen type IV) and incu-

bated overnight at 4°C. The protein solution was removed

and wells washed twice with PBS. Then, FBS (200 ll of a2% solution in DMEM) was added to the wells to block

unoccupied sites and to prevent nonspecific binding of the

bacteria. The FBS was removed using a pipette after incu-

bation at 37°C for 1 h. Two hundred microlitres of the ini-

tial bacterial suspension in DMEM was added to the ECM-

coated cell culture plates. The plates were centrifuged at

2000 g for 120 s and incubated at 37°C for 1 h. The

unbound bacteria were removed with a pipette into an Ep-

pendorf tube diluted in TBE buffer and the cell count per-

formed with a BD FACS cell sorter (USA). The number of

nonadherent and adherent bacteria was established and

expressed as a percentage of the initial bacterial suspension.

Each determination was performed in triplicates. Probiotic

strain Lactobacillus acidophilus CNRZ 1923 and Lactobacil-

lus rhamnosus GG were used as controls.

Determination of bacteriocin production and

antimicrobial spectrum

The preparation of the cell-free culture supernatant was

carried out, as described by Cocolin et al. (2007). The

strains Ent. faecium CM4 and 2CM1 were grown in MRS

broth (Oxoid) at 37°C for 18–24 h. Cultures were centri-


K. Banwo et al. Enterococcus faecium strains from milk

fuged at 13 000 g for 15 min at 4°C (Eppendorf 5810 R,

California, USA), and supernatants were collected and

adjusted to pH 7 with NaOH and filtered through a 0�2-lm membrane filter (Millipore, Billerica, MA, USA). The

antimicrobial spectrum of the cell-free culture supernatant

was carried out using agar well diffusion assay, as

described by Schillinger and Lucke (1989). The indicator

strains were grown in appropriate media such as MRS

(Oxoid) for LAB and brain heart infusion agar (Difco) for

non-LAB. Wells were punctured in the soft agar with a

sterile cork borer (5 mm) after solidification. These wells

were inoculated with 100 ll of the cell-free supernatants ofthe Ent. faecium strains, and the assay plates were incu-

bated at 30–37°C, aerobically for non-LAB and anaerobi-

cally for LAB for 18–24 h. At the end of the incubation

period, the inhibition zones were detected by a clear region

around the well. MRS broth without micro-organisms was

used as a control. Each determination was carried out in

duplicates.

Effect of enzymes, heat and pH on bacteriocin activity

The stability of the bacteriocins was tested after treatment

with catalase, proteinase K, trypsin, pepsin, lysozyme,

pronase E, lipase and a-amylase (Sigma-Aldrich). The

enzymes were added to 1 ml of neutralized cell-free cul-

ture supernatant to a final concentration of 2 mg ml�1 in

all cases, and 100 ll was inoculated into assayed plates

(Cocolin et al. 2007). The plates were incubated at 37°Cfor 18–24 h, and the antimicrobial activity spectrum was

carried out by measuring the zones of inhibition against

indicator strains Bacillus cereus DSM 2301 and Bacillus

subtilis ATCC 6633. The untreated cell-free supernatants

were used as controls. Each determination was carried

out in duplicates.

To determine the effect of pH and heat treatment on

the bacteriocin activity, final pH values of the cell-free cul-

ture supernatants, ranging from 4�0 to 9�0, were prepared,using 1 mol l�1 NaOH and HCl, and the bacteriocins

were treated at different temperatures and time: 121°C for

15 min, 100°C for 10 min and 30 min, 60°C for 10 min

and 30 min and 45°C for 10 and 30 min (Cocolin et al.

2007). The plates were incubated at 37°C for 18–24 h,

and the antimicrobial spectrum of activity was carried out

by measuring the zones of inhibition against indicator

strains B. cereus DSM 2301 and B. subtilis ATCC 6633.

The untreated cell-free supernatants were used as controls.

Each determination was carried out in duplicates.

Partial purification of the bacteriocin

For partial purification of the bacteriocins, the modified

method of Callewaert et al. (1999) and Strompfova and

Laukova (2007) was employed. The cultures were grown

for 18–24 h at 37°C, and the supernatant was obtained

by centrifugation at 10 000 g at 4°C for 15 min. The pH

was adjusted to pH 7�0 with 1 mol l�1 NaOH and the

supernatant cooled in an ice-water bath. Ammonium sul-

fate was added to the supernatant to a final saturation of

60% and held overnight at 4°C with slow and continuous

stirring on a magnetic stirrer. The suspension was centri-

fuged at 5500 g for 30 min at 4°C (Eppendorf centrifuge

5415 R), and the precipitate (surface pellicle) was resus-

pended in 50 mmol l�1 sodium phosphate buffer (pH

6�5). The dissolved precipitate was desalted by dialysis for

24 h at 4°C against 50 mmol l�1 sodium phosphate buf-

fer (pH 6�5) with two changes of buffer over a magnetic

stirrer. The molecular weight cut-off of the membrane

used was 1000 Da. The dialysate was resuspended in ster-

ile Milli Q water and frozen at �20°C until use. The

inhibitory activity of the partially purified bacteriocin was

quantified using a modified method of Yamamoto et al.

(2003) and Prasad et al. (2005). This was performed by

the preparation of 10 ll of twofold serial dilution of the

partially purified bacteriocin in sterile Milli Q water that

was spotted onto the inoculated lawn of the indicator

strain (B. cereus DSM 2301) and incubated at 30°C for 18

–24 h. The bacteriocin titre was expressed as reciprocal of

the highest dilution that exhibited inhibition. The activity

was calculated in arbitrary units per ml (AU ml�1) using

the formula: 1 AU ml�1 = 2n 9 (1000 ll per 10 ll�1),

where AU ml�1 is the arbitrary unit per ml and n is

reciprocal of the highest dilution that gave inhibition.

PCR amplification of the enterocin genes

Genes encoding for the bacteriocins were targeted by

PCR using the primers and conditions described by De

Vuyst et al. (2003) and Cocolin et al. (2007). The specific

primers for enterocins A and B were used in the amplifi-

cation. For enterocin A, the forward primer was made up

of EntFa (5′ GGT ACC ACT CAT AGT GGA AA 3′) andreverse primer EntFb (5′ CCC TGG AAT TGC TCC ACC

TAA 3′). PCR amplification conditions were as follows:

initial denaturation for 5 min at 95°C, 30 cycles of dena-

turation for 30 s at 95°C, annealing at 58°C for 30 s and

extension at 72°C for 30 s and a final elongation step of

5 min at 72°C.For enterocin B, the forward primer was made up of

Ent1 (5′ CAA AAT GTA AAA GAA TTA AGT ACG 3′) andreverse primer Ent2 (5′ AGA GTA TAC ATT TGC TAA

CCC 3′). PCR amplification conditions were as follows:

initial denaturation of 5 min at 95°C, 30 cycles of denatur-ation at 95°C for 30 s, annealing at 56°C for 30 s,

extension at 72°C for 30 s and final extension at 72°C for

5 min.



The PCR products were subjected to electrophoresis

using a 1�8% agarose gel and 19 TBE buffer containing

1 lg ml�1 ethidium bromide (Sambrook et al. 1989).

Results

Identification of the bacteriocin-producing strains

The bacteriocin-producing strains Enterococcus faecium

CM4 and Ent. faecium 2CM1 were characterized to be

Gram-positive, cocci-shaped, catalase-negative bacteria

that did not produce gas from glucose and grew at pH

9�6. Further confirmation was carried out by 16S rRNA

sequencing as stated in the Materials and methods. The

sequences are deposited in the GenBank database under

the following accession numbers JN104687 and

JN104688, respectively.

Acidification

The strain Ent. faecium CM4 reduced the pH to 4�05,while Ent. faecium 2CM1 reduced to pH 3�85 after 48-h

incubation. The initial pH of the medium was 6�54. Thestrains showed strong acidification properties by lowering

the pH of the MRS medium to <5.

Bile salts resistance

Enterococcus faecium 2CM1 displayed a growth of 79�13and 69�1%, while Ent. faecium CM4 exhibited a growth

of 74�3 and 61�67% at 0�3 and 1% bile salts concentra-

tion, respectively. However, the probiotic strain Lactoba-

cillus acidophilus CNRZ 1923 that was the control had a

growth of 77�83 and 70�1% in the presence of bile salts at

0�3 and 1% concentrations, respectively (Fig. 1). It was

observed that the Ent. faecium strains in this study gener-

ally gave good results as the probiotic strain tested.

Bile salt hydrolytic, haemolytic activity and gelatinase

characteristics

In this study, deconjugation activity was observed on

sodium glycodeoxycholate (GCDA) and TDCA, there was

no haemolytic activity and gelatinase in the plates screen-

ing tests for the selected Enterococcus faecium strains

(Table 1).

Antibiotics susceptibility profile of the strains

Enterococcus faecium CM4 demonstrated high susceptibil-

ity to vancomycin but was moderately susceptible to

streptomycin, chloramphenicol, gentamicin, pefloxacin,

ciprofloxacin and tetracycline. Enterococcus faecium 2CM1

exhibited resistance to pefloxacin, moderate susceptibility

to seven antibiotics including streptomycin, gentamicin,

tetracycline and vancomycin. There was no resistance to

clinically relevant antibiotics in the study (Table 2).

In vitro adherence assay

The strain Ent. faecium CM4 exhibited the highest value

of 38�3%, while Ent. faecium 2CM1, the least value of

37�2% for the adhesion to human fibronectin. For human

plasma fibrinogen, Ent. faecium CM4 displayed the value

of 29�9% and Ent. faecium 2CM1 value of 28�9%. The

strain Ent. faecium CM4 had the value of 32�1% and

Ent. faecium 2CM1 exhibited the value of 36�5%for human collagen type IV. The reference strains

0

10

20

30

40

50

60

70

80

90

L.acidophilus CNRZ1923

E. faecium 2CM1E. faecium CM4

Strains

Gro

wth

(%

)

Figure 1 Bile salts tolerance of the

bacteriocin-producing Enterococcus faecium

strains and reference probiotic strain

Lactobacillus acidophilus CNRZ 1923 to 0�3,0�5 and 1% bile salts concentration. Bars

represent the mean and standard deviations

of three independent experiments. ( )

0.30%; ( ) 0.50% and ( ) 1.00%.



demonstrated higher values than the selected strains for

human plasma fibrinogen and human collagen type IV,

while the Ent. faecium strains exhibited higher value for

human fibronectin (Fig. 2).

Detection of bacteriocin produced by Enterococcus

faecium CM4 and 2CM1 and the antimicrobial

spectrum

The determination of the spectrum of activity of the

bacteriocin produced by the Ent. faecium strains consid-

ered in this study, using LAB and non-LAB as indicator

organisms, is shown in Table 3. Both strains did not

show any antibacterial activity towards the representa-

tive LAB strains Enterococcus faecium ATCC 6057, Ped-

iococcus acidilactici ATCC 8042, Lactobacillus fermentum

ATCC 9338, Lactobacillus plantarum ATCC 14917, Lac-

tobacillus pentosus JCM 1558, Lactobacillus casei CMCC

1.539 and Leuconostoc dextranicum CMCC 181, while

activity was observed against Pediococcus pentosaceus

ATCC 25745. In the non-LAB strains, activity was

observed in Bacillus cereus DSM 2301, Bacillus subtilis

ATCC 6633, Micrococcus luteus and Listeria monocytoge-

nes for both strains.

Effect of enzymes, pH and temperature on bacteriocin

activity

On treatment with enzymes, the cell-free culture super-

natant demonstrated that the inhibitory activity of bac-

teriocin produced by Ent. faecium CM4 and

Ent. faecium 2CM1 was completely eliminated by pro-

teinase K and pronase E, while some activity was

observed to be retained for Ent. faecium CM4 on treat-

ment with trypsin, as shown in Tables 4 and 5. How-

ever, the activity was not affected by treatment with

catalase, pepsin, a-amylase, lysozyme and lipase for both

strains. The inhibitory activity of both Ent. faecium

strains was retained after exposure to a pH range of

4–9. The inhibitory activity of both Ent. faecium strains

Table 1 Bile salt hydrolase (BSH), Haemolytic and gelatinase

activities

Strains

BSH

Haemolysis GelatinaseGCDA TDCA

Enterococcus faecium

CM4

+ + c �

Ent. faecium 2CM1 + + c �

GCDA, glycodeoxycholic acid; TDCA, taurodeoxycholic acid; +: posi-

tive/precipitation of bile salts deconjugation; �/c: negative/no hae-

molysi.

Table

2Antibiotics

suscep

tibility

profile

oftheEn

terococcusfaecium

strains

Antibiotics

Strains

Amoxycillin

Ofloxacin

Streptomycin

Chloramphen

icol

Cefriazone

Gen

tamicin

Pefloxacin

Cotrim

axazole

Ciprofloxacin

Erythromycin

Tetracycline

Van

comycin

Ent.faecium

CM4

+(9)

�++(13)

++(12)

+(5)

++(14)

++(13)

+(5)

++(14)

�++(14)

+++(18)

Ent.faecium

2CM1

+(5)

+(7)

++(11)

++(14)

+(9)

++(14)

�++(13)

++(13)

++(12)

++(13)

++(14)

+++(15–2

0mm):highly

suscep

tible;++(10–1

4mm):moderatelysuscep

tible;+(1–9

mm):suscep

tible;�:

resistan

ce;Su

scep

tibility:presence

ofazoneofinhibition(m

m);Resistance:ab

sence

of

azoneofinhibition(m

m).



was relatively stable against heat at 45–60°C (for 10 and

30 min), respectively, and 100°C for 10 min, while its

activity was lost after treatment at 100°C for 30 min and

121°C for 15 min.

Effect of different treatments on the partially purified

bacteriocin

The activity of the control supernatants of the partially

purified bacteriocins from the two Ent. faecium strains

was determined to be 6400 AU ml�1. The results

obtained after treatment with enzymes indicated that the

bacteriocin activity was completely lost after treatment

with proteinase K, pronase E and trypsin for both strains.

There was no effect on the activity of the bacteriocin as

observed after treatment with catalase, lipase, lysozyme

and a-amylase. The activity was retained on treatment

with pH values 4–9 for Ent. faecium 2CM1, but half of

the original activity was observed for Ent. faecium CM4

at pH values 8 and 9. The retained activity observed

for both strains was not affected by heat treatment at

45–60°C (for 10 and 30 min), respectively, and at 100°Cfor 10 min but completely lost its activity at 100°C for

30 min and 121°C for 15 min (Table 6).

PCR amplification of the bacteriocin genes

The DNA of Ent. faecium CM4 and 2CM1 with primers

for enterocins A and B was amplified using PCR. Ente-

rocins A and B were targeted by the specific primers. The

PCR products obtained were for enterocin A, while there

was no amplification for enterocin B. The results got after

gel electrophoresis are shown in Fig. 3. The result high-

lighted here describes the presence of enterocin A struc-

tural gene in the two Ent. faecium strains studied.

0

10

20

30

40

50

60

70

E. faecium CM4 E. faecium 2CM1 L. acidophilusCNRZ 1923

L. rhamnosus GG

Strains

Adh

esio

n (%

)Figure 2 In vitro adherence of bacteriocin-

producing Enterococcus faecium strains and

reference probiotic strains Lactobacillus

acidophilus CNRZ 1923 and Lactobacillus

rhamnosus GG to immobilized fibronectin,

fibrinogen and human collagen type IV. Bars

represent the mean and standard deviations

of three independent experiments. ( )

Human Fibronectin; ( ) Human plasma

Fibrinogen and ( ) Human Collagen Type IV

Table 3 Antimicrobial activity of the bacteriocins produced by strains

of Enterococcus faecium CM4 and Ent. faecium 2CM1

Strains

Activity

CM4 2CM1

Lactic acid bacteria (LAB)

Ent. faecium ATCC 6057 ND ND

Lactobacillus casei CMCC 1�539 ND ND

Lactobacillus fermentum ATCC 9338 ND ND

Lactobacillus plantarum ATCC 14917 ND ND

Lactobacillus pentosus JCM 1558 ND ND

Leuconostoc dextranicum CMCC 181 ND ND

Pediococcus acidilactici ATCC 8042 ND ND

Pediococcus pentosaceus ATCC 25745 12�5 ± 0�13 10�3 ± 0�21Non–LAB

Bacillus cereus DSM 2301 16�4 ± 0�14 16�3 ± 0�20Bacillus subtilis ATCC 6633 18�3 ± 0�20 16�5 ± 0�20Candida albicans ND ND

Escherichia coli 0157:H7 ND ND

Micrococcus luteus 12�5 ± 0�12 10�1 ± 0�20Staphylococcus aureus ATCC 27702 ND ND

Listeria monocytogenes 13�3 ± 0�11 11�5 ± 0�20

American type culture collection (ATCC), Rockville, MD, USA; Chinese

microbiological culture collection (CMCC), Beijing, China; Deutsche

Sammlung von Mikroorganismen, Braunschweig (DSM), Germany;

Japanese collection of micro-organisms (JCM), Tokyo, Japan; Zone of

inhibition (mm); ND, not detectable.



Discussion

Adequate phenotypic and genotypic characterization was

employed in the identification of the Enterococcus faecium

strains as reported by Banwo et al. (2012). The ability of

the LAB strains to produce higher acid by stronger reduc-

tion in pH of the growth medium ascertains them as a

candidate for good starter culture fermentation process

(Kostinek et al. 2007; Oguntoyinbo 2007), and ability to

withstand bile at different concentrations satisfies the

selection as probiotic candidates (Vinderola and Reinhei-

mer 2003; Schillinger et al. 2005). Investigation on

enterococci of dairy origin states the poor acidifying

capacity of these organisms in milk with only a small

percentage of the strains showing a pH below 5�0 after

24 h of incubation at 37°C (Andrighetto et al. 2001; Sar-

antinopoulous et al. 2001). The Ent. faecium strains iso-

lated in our study were good acidifiers with reduction of

pH in the range 3�85–4�05 after 48-h incubation. An

acidifying potential in skim milk with a pH lowering to

about 4�5 after 24-h fermentation has also been reported

in Enterococcus faecalis strains isolated from artisan Italian

cheese (Giraffa 2003; Foulquie-Moreno et al. 2003).

Resistance to bile salts is an important criterion for the

selection of probiotic bacteria, which is a prerequisite for

the colonization and metabolic activity of the strain in

the small intestine of the host (Liong and Shah 2005;

Strompfova and Laukova 2007). The mean survival ability

of the tested strains to bile was observed to be quite high

comparable to the probiotic strain tested.

No haemolytic activity was observed for the Ent. fae-

cium strains. Haemolysin plays an important role in

enterococcal virulence, as it may increase the chance of

the infection (Morandi et al. 2006). Gelatinase activity

was not detected in the isolates by plate screening assay.

This agrees with the results of Mannu et al. (2003) and

Franz et al. (2001). Gel genes may be silent, and pheno-

types may be negative, even in the presence of a Gel gene

(Franz et al. 2001; Yoon et al. 2008). Enterococcus faecium

has been reported to be free of virulence determinants,

but there have been notable exceptions (Yoon et al.

2008). The gelE gene has been shown to be present more

Table 4 Effect of pH, temperature and enzyme activities on bacteriocin produced by Ent. faecium CM 4 against Bacillus subtilis ATCC 6633 and

Bacillus cereus DSM 2301

pH ZIBs ZIBc Temperature and time ZIBs ZIBc Enzymes ZIBs ZIBc

4 14�5 ± 0�12 14�0 ± 0�21 45°C for 10 min 15�0 ± 0�10 13�0 ± 0�12 Pronase E – –

6 16�5 ± 0�11 15�5 ± 0�13 45°C for 30 min 15�0 ± 0�13 13�0 ± 0�20 Trypsin <10�0 ± 0�10 –

7 16�0 ± 0�13 15�5 ± 0�10 60°C for 10 min 13�0 ± 0�10 11�0 ± 0�12 Proteinase K – –

9 12�0 ± 0�14 11�0 ± 0�12 60°C for 30 min 13�0 ± 0�20 11�0 ± 0�21 Pepsin 13�0 ± 0�22 13�0 ± 0�20100°C for 10 min 12�0 ± 0�20 11�0 ± 0�13 a-Amylase 17�0 ± 0�10 16�0 ± 0�12100°C for 30 min – – Lysozyme 16�5 ± 0�15 14�0 ± 0�12121°C for 15 min – – Lipase 16�0 ± 0�20 15�5 ± 0�15

Catalase 16�0 ± 0�15 14�0 ± 0�20

ZIBS: zone of inhibition for Bacillus subtilis ATCC 6633 (in mm); ZIBC: zone of Inhibition for Bacillus cereus DSM 2301 (in mm); –: no activity.

Data represent the mean ± SD of duplicates.

Each test was carried out at 37°C for 18–24 h.

Table 5 Effects of pH, temperature and enzyme activities on bacteriocin produced by Enterococcus faecium 2CM1 against Bacillus subtilis ATCC

6633 and Bacillus cereus DSM 2301

pH ZIBs ZIBc Temperature and time ZIBs ZIBc Enzymes ZIBs ZIBc

4 15�0 ± 0�20 15�0 ± 0�12 45°C for 10 min 14�0 ± 0�20 14�0 ± 0�10 Pronase E – –

6 16�0 ± 0�14 15�5 ± 0�21 45°C for 30 min 14�0 ± 0�10 12�0 ± 0�15 Trypsin – –

7 16�5 ± 0�10 16�0 ± 0�10 60°C for 10 min 14�0 ± 0�12 12�0 ± 0�12 Proteinase K – –

9 16�0 ± 0�12 16�0 ± 0�12 60°C for 30 min 11�0 ± 0�13 12�0 ± 0�10 Pepsin 13�5 ± 0�10 13�0 ± 0�10100°C for 10 min 11�0 ± 0�12 11�0 ± 0�10 a-Amylase 16�0 ± 0�12 15�5 ± 0�15100°C for 30 min – – Lysozyme 16�0 ± 0�15 16�0 ± 0�12121°C for 15 min – – Lipase 16�0 ± 0�10 16�5 ± 0�10

Catalase 16�0 ± 0�12 16�0 ± 0�20

ZIBS: zone of inhibition for Bacillus subtilis ATCC 6633 (in mm); ZIBC: zone of Inhibition for Bacillus cereus DSM 2301 (in mm); –: no activity.

Data represent the mean ± SD of duplicates.

Each test was carried out at 37°C for 18–24 h.



frequently in clinical isolates than in noninfectious strains

(Yoon et al. 2008). Eaton and Gasson (2001) reported

that Ent. faecium strains isolated from dairy products and

ham could test positive for one or more virulence deter-

minants. This determinant is found at similar frequencies

in the Ent. faecalis and Ent. faecium strains and appears

in all culture types, including isolated food, starter and

medical cultures (Yoon et al. 2008).

Enterococci are well known to be intrinsically resistant

to cephalosporins, b-lactams, sulfonamides and low levels

of clindamycin and aminoglycosides (Yousif et al. 2005).

Multiple resistant strains of enterococci had emerged in

the last decade, which showed resistance to tetracycline,

chloramphenicol and vancomycin (Yousif et al. 2005).

High levels of antibiotics resistance are related to a

combination of nonprescription antibiotic usage and the

circulation of resistant isolates in the environment with

limited sanitation facilities (Al-Jabouri and Al-Meshhada-

ni 1985). According to the findings of Mathur and Singh

(2005), there was a possible baseline for the reference

probiotic strain Ent. faecium 68, used as a probiotic for

man as well as for animal and as silage inoculant. This

strain isolated in the pre-antibiotic era is susceptible to

erythromycin (15 lg), streptomycin/penicillin (streptopen

35 lg), gentamicin (10 lg), chloramphenicol (30 lg) andtetracycline (30 lg). It also possesses intrinsic resistance

to kanamycin (30 lg), streptomycin (10 lg) and oxacillin

(5 lg) (Mathur and Singh 2005).

The Enterococcus strains were moderately sensitive to

streptomycin, gentamicin and ciprofloxacin. Enterococcus

faecium CM4 showed resistance to erythromycin (5 lg),which agreed with the intrinsic resistance characteristics

of reference probiotic strain of Ent. faecium 68. However,

it was observed that Ent. faecium CM4 showed high sus-

ceptibility to vancomycin, while Ent. faecium 2CM1 mod-

erately susceptible to this antibiotic. In another study,

enterococci isolated from Portuguese dairy products such

as milk and cheese were screened for gentamicin resis-

tance (Lopes et al. 2003). Although enterococci are gener-

ally regarded as being intrinsically resistant to low levels

of gentamicin, a high-level gentamicin resistance was

detected in many dairy isolates (Giraffa 2003; Hummel

et al. 2007).

Lactic acid bacteria are reported to bind to ECM such

as human collagen, fibronectin and human fibrinogen,

which may be exposed if the epithelial layer is injured

(Kapczynski et al. 2000; Lorca et al. 2002; Schillinger

et al. 2005). The ECM proteins binding to enterococci

Table 6 Effect of different treatments on the partially purified bacte-

riocin produced by Enterococcus faecium CM4 and Ent. faecium

2CM1

Treatment

Residual activity (AU ml�1)

Ent. faecium CM4 Ent. faecium 2CM1E

Control 6400 6400

Enzymes

Catalase 6400 6400

Trypsin – –

Pepsin 3200 3200

Pronase E – –

Proteinase K – –

Lipase 6400 6400

Lysozyme 6400 6400

a-Amylase 6400 6400

pH

4 6400 6400

5 6400 6400

6 6400 6400

7 6400 6400

8 3200 6400

9 3200 6400

Temperature (min)

45°C (10) 6400 6400

45°C (30) 6400 6400

60°C (10) 6400 6400

60°C (30) 6400 6400

100°C (10) 6400 6400

100°C (30) – –

121°C (15) – –

–: no activity.

Bacillus cereus DSM 2301 was used as the indicator strain.

AU ml�1: Arbitrary Unit per ml defined as reciprocal of highest

dilution showing inhibition of indicator strain 9 100.

Each test was carried out at 37°C for 18–24 h and the residual

bacteriocin activity determined.

Data represent test in duplicates (n = 2).

Figure 3 Polymerase chain reaction (PCR) products obtained from

the amplification using the specific primer for enterocins A and B.

Lane 1: molecular ladder (50 bp); lanes 2 and 3: enterocin A amplifi-

cation for Enterococcus faecium CM4 and Ent. faecium 2CM1; lanes

4 and 5: no amplification for enterocin B for Ent. faecium CM4 and

Ent. faecium, respectively.



could be involved in tissue colonization by enterococci as

shown for other Gram-positive pathogens (Wadstrom

et al. 1987; Kostrzynska et al. 1992). Our results showed

the ability of enterococci to bind to fibronectin at 30% in

contradiction with the findings of Zareba et al. (1997)

who reported that enterococci are nonbinders of rela-

tively low binding capacities of 5% to 7%. The strongest

binding capacities were observed in the human fibronec-

tin, but binding to human collagen type IV and fibrino-

gen was lower for the strains tested. As reported by

Morelli (2000), adherent strains of probiotic bacteria are

more likely to stay longer in the intestinal tract and

therefore may have a better opportunity to exhibit meta-

bolic and immunomodulatory effects than the nonadher-

ing counterparts. The enterococci exhibited sufficient

binding rate for probiotics. Moreover, as reported by

Strompfova and Laukova (2007), probiotic strains that

may not be able to show in vitro and/or in vivo adhesion

characteristics can still show positive effects in the hosts.

In addition, the rate of equality of in vitro and in vivo

capabilities is also questionable.

Numerous strains of enterococci associated with food

systems, mainly Ent. faecium and Ent. faecalis, are capable

of producing a variety of bacteriocin called enterocin

with broad spectrum activity (Franz et al. 1996; Ennahar

et al. 2001; Giraffa 2003). Enterocins usually belong to

class II bacteriocins, which are small, heat stable and

nonlantibiotics (Giraffa 2003). In this study, two Entero-

coccus strains isolated from cow’s milk show antimicro-

bial activity against Gram-positive bacteria, including

Bacillus cereus DSM 2301, Bacillus subtilis ATCC 6633,

Micrococcus luteus, Listeria monocytogenes and Pediococcus

pentosaceus ATCC 25745. The inhibitory activity of the

bacteriocin produced by the Ent. faecium strains com-

prises Gram-positive bacteria including List. monocytoge-

nes but showed limited or no activity against Gram-

negative indicators. This observation is in agreement with

previous workers on enterocins (Cocolin et al. 2007;

Javed et al. 2011). Enterococcal bacteriocins that show

antilisterial activity are of great importance in food and

dairy industries (Rodriguez et al. 2000; De Vuyst et al.

2003). Olasupo et al. (1999) isolated a bacteriocin-pro-

ducing Ent. faecium NA01 strain from wara, a fermented

skimmed cow milk product from West Africa. Enterocins

characterization and screening from Ent. faecium of dif-

ferent origins has been an important area of research, to

enhance the safety of this strain as starter culture for food

products (Foulquie-Moreno et al. 2003; Cocolin et al.

2007).

The effect of pH on enterocin production and appar-

ent degradation was as intense as the effect on bacterial

growth (Foulquie-Moreno et al. 2003). This effect was

similar in our study for Ent. faecium CM4, which

expressed a reduction in activity at pH 8 and 9 and still

had a very good activity at pH 4–7; however, Ent. fae-cium 2CM1 exhibited good activity. This agrees with the

work of Parente and Ricciardi (1999), who reported the

bacteriocin activity for Enterocin 1146, which displayed a

decrease in the early stationary phase at pH values higher

than 4�5. The decrease of activity was ascribed to the

adsorption of bacteriocin molecules on the cell surface of

producer cells; and this depends on the pH of cell envi-

ronment being more pronounced at higher pH (Parente

and Ricciardi 1999; Leroy and De Vuyst 2003; Vizoso-

Pinto et al. 2006). The bacteriocins produced lost its

activity after exposure to heat at 100°C for 30 min but

still had full activity at 100°C for 10 min. This indicates

sensitivity to heat, which was also reported by Du Toit

et al. (2000) and Cocolin et al. (2007) who observed that

Enterocin 1170 was sensitive to heat as its activity

decreased at 80 and 100°C after 30 min, and the activity

was retained after 100 and 60°C for 10 min for Ent. fae-

cium M241 and M249, respectively. pH values affecting

the activity of enterocins produced are in accordance

with those reported by Du Toit et al. (2000). The activity

of Enterococcus over a wide range of pH may be advanta-

geous when produced in the gastrointestinal tract, where

pH levels are known to vary from pH 3 in the stomach

to >pH 7�0 in the large intestine as reported by Du Toit

et al. (2000) and Strompfova and Laukova (2007).

The effect of enzyme was investigated on the bacterio-

cin produced; it was observed that the activity of the

enterocin was completely lost after the action of protein-

ase K and pronase E and trypsin for Ent. faecium 2CM1.

This indicates that the bacteriocin produced is proteina-

ceous in nature. The ability to retain some activity after

treatment with trypsin in Ent. faecium CM4 and pepsin

for both strains agrees with the work of Cocolin et al.

(2007), in which some activity was retained for strains

Ent. faecium M241 and M249 after treatment with pep-

sin. This is in agreement with report of other works on

enterocin production by Ent. faecium strains (Cocolin

et al. 2007; Strompfova and Laukova 2007). However, the

enterocins produced in our study retained its full activity

on treatment with a-amylase, lysozyme, lipase and cata-

lase and reduced activity for pepsin. The neutralized cul-

ture supernatants were not inactivated by lysozyme and

lipase, which suggest that the Enterococcus in this study

do not require a lipid or carbohydrate moiety for activity

as also reported by Du Toit et al. (2000).

The genetic determinants of the bacteriocin genes for

the bacteriocin-producing organisms were targeted for

enterocins. The primer specific for enterocins A and B

were used as described by De Vuyst et al. (2003) and

Cocolin et al. (2007). The PCR product of the expected

size for the enterocin produced by the bacteriocin genes



was observed to be about 140 bp for enterocin A. This is

in agreement with the size observed also by Cocolin et al.

(2007). After amplification, it was confirmed that the

bacteriocin-producing Enterococcus species (Ent. faecium

CM 4 and Ent. faecium 2 CM1) had the presence of

enterocin A but not enterocin B. In the works of Cocolin

et al. (2007), there was the presence of both enterocins A

and B in Ent. faecium M 241 and Ent. faecium M 249.

The structural gene of enterocin A is widely distributed

among Ent. faecium strains, whereas that of enterocin B

always occurs in the presence of enterocin A as reported

by De Vuyst et al. (2003). This implies that there are no

transport genes for enterocin B production as reported

by Franz et al. (1999) in De Vuyst et al. (2003). The

authors suggested that these genes may be lost due to

mutations, which could mean that the structural gene of

enterocin B was lost by several strains, resulting in the

observation that the structural gene of enterocin A occurs

solely in some strains (De Vuyst et al. 2003). The bacte-

riocin genes were observed to be chromosomal because

there was no plasmid isolated from the Ent. faecium

strains (results not shown). This indicates that the genes

for bacteriocin activities are located on the chromosomal

DNA. This is in agreement with the work of De Vuyst

et al. (2003); they further stated that chromosomal locali-

zation of enterocin genes can be an intrinsic characteris-

tic, while plasmid localization is a result of acquired

antibacterial potential through conjugal gene transfer.

The application and importance of bacteriocin-produc-

ing Ent. faecium strains is of immense usefulness in food

and dairy industries (Giraffa 2003; Cocolin et al. 2007).

The use of these strains as starter cultures, cocultures or

protective cultures is becoming an important aspect that

can lead to the production of safer food (Knorr 1998;

Giraffa 2003; Cocolin et al. 2007). The use as a coculture

was addressed by Cocolin et al. (2007) who cocultured

the bacteriocin-producing Ent. faecium M 241 and

Ent. faecium M 249 in skimmed milk with Lact. monocyt-

ogenes. Both enterococci strains studied by the authors

showed a notable inhibition towards List. monocytogenes

with delay in growth of the pathogen (Cocolin et al.

2007). In this study, the enterocins produced by the

strains of Enterococcus faecium showed considerable inhi-

bition against List. monocytogenes. It has also been

reported that enterocin-producing strains of Ent. faecium

is of great potential in dairy technology (Giraffa 2003).

Therefore, enterocins or the producers show a potential

for a diary application as biopreservatives or protective

cultures as reported by Cocolin et al. (2007).

In conclusion, the Ent. faecium strains exhibited a good

probiotic activity because of their tolerance to low pH,

BSH activity, safety and in vitro adherence properties and

the enterocins produced displayed inhibitory activities

against food pathogens and spoilage micro-organisms.

The use of bacteriocin-producing Enterococcus strains as

starter cultures, protective agents in fermented foods and

as probiotics is strain specific as reported by De Vuyst

et al. (2003). The authors also reiterated that it is safer to

use Ent. faecium strains than Ent. faecalis strains. The

bacteriocin produced by the strains demonstrates their

ability to be included in a starter culture for food and

dairy fermentation.

Acknowledgements

The award of a postgraduate fellowship by CAS-TWAS to

Kolawole Banwo for the research period at Institute of

Microbiology, Chinese Academy of Sciences, Beijing,

China, is gratefully acknowledged. The authors appreciate

Dr. Jin Zhong for kindly providing some of the reference

strains used in this study.

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