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Mohammed et al, 2013..

Influence of Nutrient Supplements on Bacteriocin Production by Lactobacillus bulgaricus

Journal of Engineering & Applied Sciences Vol. 2(1), pp10-15, July, 2013
International Research Journal Group
All Rights Reserved
Available online http://www.irjset.com

Influence of Nutrient Supplements on Bacteriocin


Production by Lactobacillus bulgaricus Y34 and
Lactococcus lactis N22
*1Mohammed,S.S.D., 1Bala, E, 2Yakubu, A, 4Oyewole, O.A, 3Garba, Y and 1Muhammad, I. L.
1

Department of Microbiology, Ibrahim Badamasi Babangida University, Lapai, Niger State, Nigeria
Department of Biochemistry, Ibrahim Badamasi Babangida University, Lapai, Niger State, Nigeria
3
Department of Crop Production, Ibrahim Badamasi Babangida University, Lapai, Niger State, Nigeria
4
Department of Microbiology, Federal University of Technology, Minna, Niger State, Nigeria
2

Abstract The influence of nutrient supplements on bacteriocin production by Lactobacillus bulgaricus, Y34 and Lactococ-

cus lactis N22 was carried out. The supplements of nutrients with De Man Rogosa Sharpe (M.R.S) broth demonstrated that
large quantities of bacteriocins could be produce by addition of glucose (1%), Nacl (1 and 2%), beef extract (2%), yeast extract (1, 2 and 3%), tween 80 (0.5 and 1%) while the addition of 0.1% and 0.5% triammonum citrate, 0.5 and 1% sodium acetate, magnesium sulphate, manganese sulphate and potassium phosphate had effects on the bacteriocin production. Bacteriocin
produced by L. bulgaricus. Y34 and L. lactis N22 had large spectrum of inhibition against the pathogenic, food spoilage microorganisms (indicator microorganisms) employed as test strains. The bacteriocins produced by test strains inhibited the
growth of indicator microorganisms i.e Staphylococcus aureus N23, Salmonella typhimurium N17, Bacillus cereus N12,
Shigella dysentriae N25, and Pseudomonas aeruginosa N1. L. bulgaricus Y34 had the highest bacteriocin activity of
600000.0 AU/mL while L. lactis N22 had 580000.0 AU/mlL as it highest bacteriocin activity. Maximal bacteriocin production was achieved at pH between 3.9 and 4.5 and incubation period of 48 hours at temperature of 30oC for the two species of
lactic acid bacteria used in this study.
Keywords: Bacteriocins, Lactic acid bacteria, De Man Rogosa Sharpe broth, indicator microorganisms, Nutrient supplements.
* Corresponding author:
mosada78@yahoo.com (Mohammed,S.S.D)
Published online at http:/www.irjset.com
Copyright 2013 International Research Journal Group

1. Introduction

Lactic and bacteria are described as microaerophilic as


they do not utilize oxygen (Adam and Moss, 1995). Lactobacilli are important organisms recognized for their fermentative ability as well as their health and nutritional benefits
(Gilliland, 1990). LAB is often inhibitory to other microorganisms and this is the basis of their ability to affect the
keeping quality and safety of many food products. The principal factors which contribute to this inhibition are low pH,
organic acids hydrogen peroxide, ethanol, nutrient depletion,

low redox potential and bacteriocin production (Adams and


Nicolaides, 1997).
Bacteriocins are proteinaceuous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial
strain (Gratia, 2000). Devugst and vanclamme (1994) worked
on production of bacteriocin (nisin) using a specie of lactic
acid bacteria (Lactococcus lactis Subsp lactis) and reported
that nisin was the first bacteriocin used on commercial scale
as food preservative date back to the first half of the century.

Mohammed et al, 2013..Influence of Nutrient Supplements on Bacteriocin Production by Lactobacillus bulgaricus

Though several types of bacteriocins from food-associated


lactic acid bacteria have been identified and characterized, of
which the important ones are diplococcin, acidophilin, bulgarican, helveticin, lactacin, plantaricin and nisin (Nettles and
Barefoot, 1993). Maximal bacteriocin production could be
obtained by supplementing a culture medium with growth
limiting factors. Such as Sugars, Vitamins and Nitrogen
Sources, by regulating pH or by choosing the best-adapted
culture medium (Vignolo et al., 1995). For the benefit of this
study, the effects of nutrient supplementation on bacteriocin
production by some species of LAB is the sole aim of this
research.

2. Material and methods


2.1 Collection of Samples: Five (5) samples of Yoghurt
and Nono were obtained from Bosso Market-Minna. The nono
samples were collected in a sterile sample bottles and transferred to Federal University of Technology Minna, Microbiology Laboratory for Isolation of Lactic acid bacteria and
indicator microorganisms.
2.2 Isolation lactic acid bacteria and indicator microorganisms: The pour plate methods of Fawole and Oso.
(1998)., Cheesbrough. (2003)., Oyeleke and Manga. (2008)
were followed for the isolation of LAB and indicator microorganisms from the fermented milk products (yoghurt and
nono).
2.3 Characterization of and Identification of Microbial
Isolates: Microbial isolates were characterized and identified
based on colony morphology, cell morphology and biochemical tests described by Fawole and Oso. (1998).,
Hammes et al.(1999)., Cheesbrough. (2003)., Oyeleke and
Manga (2008).
2.4 Effects of nutrient Supplements on the production of
bacteriocins: The effects of nutrient supplements on bacteriocin production by Lactobacillus bulgaricus Y34 and Lactococcus lactis N22 was evaluated according to the procedure of Ogunbanwo et al. (2003) by incorporating each of the
following salts in to De Man Rogosa Sharpe (MRS) Medium.
Glucose (1.0 and 2.0%), NaCl (1.0,2.0 and 3.0%), beef extract
(1.0 and 2.0%), yeast extract (1.0,2.0 and 3.0%), Tween 80
(0.5 and 1.0%), Triammonium citrate (0.1 and 0.5%),
MnSO4 .4H2O (0.1 and 0.5%), K2HPO4 (0.1 and 0.5%), Sodium acetate (0.5 and 1.0%), and MnSO4. 7H2O (0.5 and
1.0%).
2.5 Bacteriocin Assay (production): Lactobacillus bulgaricus Y34 and Lactococcus lactis N22 were propagated in
1000ml of De Man Rogosa Sharpe (MRS) broth supplemented
with the nutrients (pH 5.8) at 30oC for 48 hours for each of the
isolates respectively. For the extraction of bacteriocins, a cell
free solution of bacteriocins was obtained by centrifugation
for 20 minutes. The cultures were then adjusted to pH 7.0
using 1m NaOH to exclude the antimicrobial effects of organic acid, and then followed by filtration of the supernatant.

Through 0.2 pore size cellulose acetate filter (Schillinger and


lucke, 1989) to obtain crude bacteriocin for each isolate respectively. Inhibition activity from hydrogen peroxide (H2O2)
was eliminated by the addition of 5mg/ml catalase (Daba et al.,
1991). The broth culture obtained was tested for pH, growth of
producer organisms and bacteriocin activity against the indicator microorganisms (Staphylococcus aureus N23, Salmonella typhimurium N17, Bacillus cereus N12, Shigella
dysenteriae N25 and Pseudomonas aeruginosa N1) has described by Brinkten et al. (1994), Graciela et al. (1995) and
Ogunbanwo et al. (2003).

3. Results

Lactobacillus bulgaricus Y34 and Lactococcus lactis N22


were observed to be good bacteriocin producers after propagating them in De Man Rogosa Sharpe broth supplemented
with some nutrients at different concentrations. The influence
of nutrient supplementation on bacteriocin production by L.
bulgaicus Y34 showed that large amount of bacteriocin was
synthesized only when the medium (MRS broth) was supplemented with 1% glucose, 1% and 2% NaCl, 2% beef extract, 1, 2 and 3% yeast extract, 0.5 and1% Tween 80. While
addition of 0.5% and 1% of triammonium citrate, manganese
sulphate, potassium hydrogen phosphate, sodium acetate and
magnesium sulphate caused a decrease in bacteriocin production after testing the bacteriocin culture for bacteriocin activity
against the indicator microorganisms. The decrease observed
in bacteriocin production might be due to the effects of the
supplemented salts on the test isolates (Table1). Similarly the
influence of nutrient supplements on bacteriocin production
by L. lactis N22 showed that large amount of bacteriocin was
synthesized only when the MRS medium was supplemented
with 1% glucose, 1 and 2% Nacl, 1and 2% beef extract, 1,2
and 3% yeast extracts, 0.5, 1% Tween 80, and 0.1% triammoniun citrate (Table 2). While the supplementation of the
MRS broth with 0.5% and 1% of triammonium citrate, manganese sulphate, potassium hydrogen sulphate, sodium acetale
and magnesium sulphate cause a decrease in bacteriocin
production and this might be due to the reaction of the test
microbes to the salt supplementation (Table 2). L. bulgaricus
Y34 had producing ability of between 0.40 to 0.92, pH of 3.92
to 4.20 and bacteriocin activity (AU/mL) of 3000+0.00 to
60000.00 while L. lactic N22 had producing ability between
0.40 to 6.04, pH of 3.03 to 4.90 and bacteriocin activity ( AU
/mL) of 2900 +0.00 to 58000.00 (Table 1 and 2). L. bulgaricus Y34 had the highest bacteriocin activity of 600000.00
AU /mL while L. lactis N22 had bacteriocin activity of
58000.00 AU/mL (Table 1 and 2). The inhibition of
Staphylococcus aureus N23, Salmonella typhimurium N17,
Bacillus circus N12, Shigella dysentriae N25 and Pseudomonas aeruginosa N1 was observed (+6mm zone of indicator)
after testing them against the most effective bacteriocin,
produced by L. bulgaricus Y34 and L. lactic N22 (Table 3).

Mohammed et al, 2013..Influence of Nutrient Supplements on Bacteriocin Production by Lactobacillus bulgaricus

Table 1. Effect of nutrient supplements on bacteriocin production by Lactobacillus bulgaricus Y34.


Medium composition

Quantity of
Nutrient used (%)

Growth
(580nm)

pH of
medium

Bacteriocin
activity (Au/ml)

MRS (unsupplemented)

0.0

0.90

3.91

5800 0.00*

MRS + glucose

1.0

0.90

4.07

6000 0.00*

2.0

0.92

4.09

3000 0.00

1.0

0.70

3.92

6000 0.00*

2.0

0.73

3.97

5800 0.00*

3.0

0.60

3.95

3000 0.00

1.0

0.90

3.93

3000 0.00

2.0

0.92

3.98

6000 0.00*

1.0

0.92

4.03

5800 0.00*

2.0

0.90

4.07

6000 0.00*

3.0

0.89

4.20

6000 0.00*

0.5

0.70

4.01

5800 0.00*

1.0

0.71

4.00

6000 0.00*

MRS + Triammonium

0.1

0.40

4.01

3000 0.00

Citrate

0.5

0.70

MRS + Nacl

MRS + Beef extract


MRS + yeast extract

MRS + Tween 80

MRS + MnSo4.4H2

MRS + K2 HPO4
MRS + Sodium acetate

3000 0.00

0.1

0.89

4.93

3000 0.00

0.5

0.70

4.00

3000 0.00

0.1

0.50

4.01

3000 0.00

0.5

0.80

0.04

3000 0.00

0.50

3.98

3000 0.00

0.60

4.01

3000 0.00

4.06

3000 0.00

0.5
1.0

MRS + MgSO4 7H2O

4.06

0.5
1.0

0.90
0.60

4.00

3000 0.00

Growth, pH and bacteriocin determinations were done after 48 hours of incubation. *:potential bacteriocins produced.
MRS: Deman Rogosa Sharpe medium; AU/mL: Activity per milliliter; nm: nanometer Y: Yoghurt
Table 2. Effects of nutrient Supplements on bacteriocin production by Lactococcus lactis N 22
Medium composition

Quantity of
Nutrient used (%)

Growth

pH of

(580nm)

medium

Bacteriocin
activity (Au/ml)

MRS (unsupplemented)

0.0

0.90

3.72

5600 0.00*

MRS + glucose

1.0

0.80

4.03

5800 0.00*

2.0

0.72

4.07

2900 0.00

1.0

0.70

3.84

5800 0.00*

2.0

0.70

3.90

5600 0.00*

3.0

0.60

3.95

2900 0.00

1.0

0.90

3.93

5800 0.00*

MRS + Nacl

MRS + Beef extract

Mohammed et al, 2013..Influence of Nutrient Supplements on Bacteriocin Production by Lactobacillus bulgaricus

2.0
MRS + yeast extract

0.89

3.90

5800 0.00*

1.0

0.94

3.03

5800 0.00*

2.0

0.90

4.07

5800 0.00*

3.0

0.90

4.50

5800 0.00*

0.5

0.70

4.02

5600 0.00*

1.0

0.60

3.09

5800 0.00*

MRS + Triammonium

0.1

0.40

3.07

5600 0.00*

Citrate

0.5

0.70

4.06

2900 0.00

MRS + MnSO4.4H2O

0.1

0.89

4.90

2900 0.00

MRS + Tween 80

0.5

0.80

4.00

2900 0.00

MRS + K2 HPO4

0.1

0.70

3.09

2900 0.00

0.5

0.90

4.06

2900 0.00

MRS + Sodium acetate

0.5

0.50

3.90

1.0

0.60

4.01

2900 0.00

0.5

0.90

3.06

2900 0.00

1.0

0.60

4.00

2900 0.00

MRS + MgSO4 7H2O

2900 0.00

Growth, pH and bacteriocin determinations were done after 48 hours of incubation. *:potential bacteriocins produced.
MRS: Deman Rogosa Sharpe medium; AU/mL: Activity per milliliter; nm: nanometer Y: Yoghurt

Table 3 Inhibition of indicator microorganisms by bacteriocins produced by Lactobacillus bulgaricus Y34 and Lactococcus lactis N22 after nutrient Supplements of MRS
Indicator microorganisms

Source

Zone of inhibition (mm) caused by bacteriocins produced by


Lactobacillus bulgaricus Y34

Lactococcus lactis N22

Staphylococcus aureus N23

Nono

+(6mm)

+(6mm)

Salmonella typhimurium N17

Nono

+(6mm)

+(6mm)

Bacillus cereus N 12

Nono

+(6mm)

+(6mm)

Shigella dysenteriae N 25

Nono

+(6mm)

+(6mm)

Pseudomonas aeruginosa N1

Nono

+(6mm)

+(6mm)

Key:
+:Inhibition
-:No Inhibition
N: Nono

Mohammed et al, 2013..Influence of Nutrient Supplements on Bacteriocin Production by Lactobacillus bulgaricus

4. Discussion
The present study was aimed at determining the influence of
nutrient supplements on bacteriocin production. L. burglarious Y34 and L. lactis N22 were able to produce bacteriocin
which had wide inhibitory spectrum towards the indicator
microorganisms. Thus, Variation in the concentration of constituents/supplements of cultivating media (MRS broth) has
an influence on the amount of bacteriocin produced by L.
burglaricus Y34 and L. lactis N22. The highest bacteriocin
activity of 6000+0.00 AU/mL was exhibited by L. burglarious Y34 when 2%. Glucose, 1% Nacl, 2% beef extract,1, 2
and 3% yeast extract and 1% tween 80 was added to MRS
broth. While L lactis N22 exhibited highest bacteriocin activity of 58000.00 AU/mL when 2% beef extract, 1, 2 and 3%
yeast extract, 0.5 and 1% tween 80 were added to MRS broth.
While the supplements of triammonium citrate, manganese
sulphate, potassium hydrogen sulphate, sodium acetate and
magnesium sulphate at 0.1% and 0.5% yielded no improvement in bacteriocin production. Similar observations have
been made previously by Daba et al. (1993) who reported
similar findings in the production of mensenterocin 5. Sani et
al. (1999) also reported that highest bacteriocin activity was
obtained when glucose and peptone were varied to 0.25%
and 0.5% in the constitute MRS broth. While bacteriocin
activity was not detected at 2% glucose and peptone level.
The ineffectiveness of manganese sulphate, potassium hydrogen sulphate, sodium acetate and magnesium sulphate
supplemented with the MRS broth might be due to the activity of extracellular endogenous proteinases induced during
the growth phase of the test strains. This is similar to the
findings of Paired et al. (1990) who revealed that the decrease in bacteriocin production by Lactococcus lactis CNRZ
481 after nutrient supplementation could be due to activity of
extracellular proteinases induced during the growth phase.
Some reports indicate that bacteriocin are produced throughout the experimental growth phase and note solely during late
logarithmic or early stationary phase (Joerger and Klaenhammer, 1986., Paid et al., 1990). The bacteriocin produce
by L bulgaricus Y34 exhibited bacteriocin activity between
3000+0.00 to 6000+0.00 Au/ml while the bacteriocin produced by L. lactis N22 exhibited histrionic activity between
29000.00 to 58000.00 Au/ml against Staphylococcus
aureus N23, Salmonella typhimurium N17, Bacillus cereus
N12, Shigella dysenteriae N25 and Pseudomonas aeruginosa
N1. This is in conformity with the findings of Ogumbanwo
et al. (2003) who reported the bacteriocin produced by Lactobacillus plantarium F1, and Lactobacillus brevis OG1 exhibited bacteriocin activity between 3200+0.00 to 6400+0.00
Au/ml against Escherichia coli, Listeria monocytogene and
Enterococcus faecalis. This findings also agrees with Mohammed et al.( 2012 ) who reported that pediocin and nisin
produced by Pediococcus halophilus W9 and Lactococcus
lactis N24 exhibited bacteriocin activity between 50000.00

and 56000.00 AU/mL against Pseudomonas aeruginosa,


Shigella dysenteriae and Eschericia coli.
Modification of nutrient of cultivating media should be considered for maximal production of bacteriocin that has potential use as biopreservative (Biwas et al., 1991). Therefore, the
Findings in this study revealed that bacteriocin could be
largely produced if the medium is supplemented with nutrients that could favour the growth and comfortability of the
producer organisms (LAB).

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