Food and Nutrition Sciences, 2013, 4, 342-356
http://dx.doi.org/10.4236/fns.2013.43045 Published Online March 2013 (http://www.scirp.org/journal/fns)
Study Bacteriocin Production and Optimization Using New
Isolates of Lactobacillus spp. Isolated from Some Dairy
Products under Different Culture Conditions
Hoda Mahrous1*, Abeer Mohamed2, M. Abd El-Mongy2, A. I. El-Batal3, H. A. Hamza2
1Department of Industrial Biotechnology, Genetic Engineering and Biotechnology Research Institute, Menoufiya University, Shibin
El Kom, Egypt; 2Department of Microbial Biotechnology, Genetic Engineering and Biotechnology Research Institute, Menoufiya
University, Shibin El Kom, Egypt; 3National Center for Radiation Research and Technology, Cairo, Egypt.
Received October 8th, 2012; revised January 24th, 2013; accepted February 1st, 2013
ABSTRACT
Lactobacilli belong to the group of lactic acid bacteria (LAB), that have several distinguished abilities such as produc-
tion of lactic acid, enzymes such as β-Galactosidase and natural antimicrobial substances called bacteriocins. Bacterio-
cin is a biopreservative agent potential of suppressing growth of some contaminant bacteria in food industry but its
commercial availability is limited and costly. The study aimed to select isolates of Lactobacillu s spp. potential for pro-
ducing bacteriocins to suppress the growth of Escherichia coli ATCC 25922 and Bacillus subtilis NCIB3610, and to
optimize the process of bacteriocin production. Results obtained in this study showed that L. acidophilus isolate CH1
was selected as the best candidate for bacteriocin among the four isolates that tested. The largest amounts of the bacte-
riocins were synthesized only in MRS medium was supplemented with K2HPO4 (1.0%), Tween 80 (1%), Beef extract
(1%), glucose, cyctein and peptone extract (1%). The optimization of culture conditions for bacteriocin production areas
showed that corn steep liquor medium was the best medium for all isolates against Bacillus subtilis while no effect was
observed on Escherichia coli ATCC 25922 except when used MRS medium. The optimum conditions for bacteriocin
production were pH 6.0, temperature 34˚C with 4% Phenyl acetamide showing the greatest growth inhibition areas.
Keywords: Lactobacillus acidoph ilus; Lactic Acid Bacteria; Bacteriocins Production
1. Introduction
Lactic acid bacteria (LAB) produce a number of antim-
icrobial substances such as organic acids, free fatty acids,
ammonia, reuterin, diacetyl, hydrogen peroxide and bac-
teriocin, which have the capacity to inhibit the growth of
food spoilage and pathogenic organisms [1]. Bacteriocins
are proteinaceous and ribosomally synthesized antibacte-
rial compounds produced by certain LAB during lactic
acid fermentations that exhibit bactericidal activity against
closely related species [2,3]. In recent years, a renewed
interest in bacteriocin like activities has led to the dis-
covery, isolation, and purification of bacteriocins from
both gram-negative and gram-positive bacteria. They are
now being considered for a variety of antimicrobial uses
in foods and medicine [4]. Some bacteriocins produced
by lactic acid bacteria, such as nisin, inhibit not only
closely related species but are also effective against food-
borne pathogens and many other gram-positive spoilage
bacteria [5]. For this reason, bacteriocins have attracted
considerable interest for use as natural food preservatives
in recent years, which have led to the discovery of ever
increasing potential sources of these protein inhibitors.
LAB bacteriocins are divided into three main groups,
based on their amino acid sequence, mode of action, heat
tolerance, biological activity, presence of modified amino
acids, and secretion mechanism. The classes I and II are
further divided into subgroups, and the members of these
classes are the most studied because they are so wide-
spread among the LAB and due to their heat stability.
The class III bacteriocins are heat-labile and therefore
less interesting in the terms of food processing and pro-
tection. Quite recently a new classification has been pro-
posed by Cotter et al. [6]. In this scheme the most dra-
matic change is the removal of class III bacteriocins to
their own group of “bacteriolycins”, hence making the
group of bacteriocins smaller and more strictly defined.
Lactobacillus bacteriocins are found within each of the
four major classes. Class I bacteriocins (antibiotics) were
discovered in the lactobacillaceae by Mortvedt et al. [7].
These bacteriocins are small membrane-active peptids
*Corresponding author.

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
Isolated from Some Dairy Products under Different Culture Conditions 343
(<5 kDa) containing an unusual amino acids, lanthionine.
The class II bacteriocins are small heatstable, non-lan-
thionine containing and membrane-active peptides (<10
kDa). The class III bacteriocins, have been found in
Lactobacillus, include heat labile proteins of large mo-
lecular mass. The class IV bacteriocins are a group of
complex proteins, associated with other lipid or carbohy-
drate moieties, which appear to be required for activity.
They are relatively hydrophobic and heat stable [8].
Different bacteriocin exhibits different inhibition pro-
file on food spoilage and pathogenic microorganisms.
Therefore, they could be natural replacements for syn-
thetic food preservatives [9]. In order to increase the
productivity of bacteriocins, a better understanding of
factors affecting their production is essential. Bacteriocin
production has been reported to be affected by several
factors including carbon and nitrogen sources; and fer-
mentation conditions, such as pH, temperature and agita-
tion [9].
The optimization of bacteriocin production and en-
hancement of its activity are economically important to
reduce the production cost. Thus, the aims of this study
were to formulate industrial media for bacteriocin pro-
duction by four lactic acid bacteria isolates and the opti-
mization of culture conditions for maximizing bacterio-
cin production.
2. Materials and Methods
2.1. Isolation and Identification of Lactic Acid
Bacteria
The lactic acid bacteria were isolated from raw milk and
Ras cheese, by appropriate dilutions with NaCl physio-
logical. Dilutions (10−1 - 10−6) were prepared and plated
on de Man Rogosa agar (MRS agar) medium (Hi Media
Laboratory Pvt. Ltd. Mumbai, India) to isolate the Lac-
tobacillus spp and incubated at 37˚C for 48 - 72 h [10].
The strains were sub-cultured onto MRS agar slant incu-
bated at 30˚C for 24 h and preserved in 20% glycerol at
−80˚C. One of the isolates was selected for further stud-
ies. It exhibited strong inhibitory activity against indica-
tor strains. It was identified on the basis of growth, cell
morphology, gram staining and catalase activity. Further,
identification was performed according to carbohydrate
fermentation patterns and growth at 15˚C and 45˚C in the
de Man Rogosa Sharpe (MRS) broth based on the char-
acteristics of the lactobacilli as described in Bergey’s
Manual of Determinative Bacteriology [11-14] and fer-
mentation of different carbon sources (API 50 CHL,
bioMerieux SA, France). The ability of these isolated
strains to produce acids from different carbohydrates was
determined by API 50 CHL test kit (bioMerieux SA,
France).
2.2. Selection of Isolates Producing Bacteriocin
2.2.1. Treatment of Bacteria Prior to Production of
Bacteriocin
The isolates were tested for their production of Bacterio-
cin. E. coli ATCC 25922 and Bacillus subtilis NCIB3610
were used as indicator microorganism in all assays. Indi-
cator microorganisms used are propagated for 48 h in the
Nutrient agar media, and at the temperatures indicated
30˚C.
2.2.2. Detection of Antibacterial Activity
The antimicrobial activity of the isolates during the growth
phase against Gram negative bacterium E. coli ATCC
25922 and Gram positive bacterium Bacillus sub tilis
NCIB3610 was evaluated by deferred methods: 1) well-
diffusion assay [15] and 2) Tetrazolium/formazan-test
method [16].
2.3. The Maximization of Bacteriocins
Production
2.3.1. Determination of Bacteriocin Production at
Different Culture Conditions
The effects of different temperatures and initial pH on
the bacteriocin production were tested. MRS broth (10
mL) was inoculated with each isolate and incubated at
different temperatures such as 10˚C, 20˚C, 30˚C, 40˚C
and 50˚C to study the effect of different temperatures on
the bacteriocin production. The effect of initial medium
pH on bacteriocin production was determined by adjust-
ing the MRS broth to different pH levels of 2, 4, 6, 8 and
10, respectively. Each tube was inoculated with 2.0% v/v
of an 18 h-old culture of the four isolates and incubated
at 30˚C for 96 h, without agitation.
2.3.2. Influence of Medium Component on the
Production of Bacteriocins
The effect of medium ingredients on bacteriocin produc-
tion was evaluated using composed media. The supple-
ments studied were tryptone, yeast extract, beef extract,
triammonium citrate sodium acetate, MgSO4-7H2O,
K2HPO4, NaCI, glucose and tween 80 (1%, 2% and 3%)
for each. Then, cells were removed by centrifugation at 6
000 rpm for 20 min. the culture media was adjusted to
pH 7.0 using 1 M NaOH to exclude the antimicrobial
effect of organic acids, followed by filtration of the su-
pernatant through a 0.2 ml pore-size cellulose acetate
filter.
2.3.3. Influence of Different Media on the Production
of Bacteriocins
The effect of different medium on bacteriocin production
was evaluated using media at 30˚C for 48 hours.

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
Isolated from Some Dairy Products under Different Culture Conditions
344
 Selective medium (MRS media) as control.
 Medium (A) Corn steep liquor-Lactose medium [17].
 Medium (B) Corn steep liquor-Lactose medium [18].
 Medium (D) Corn steep liquor medium [19].
 Medium (E) Glycerol-molasses-liquid medium [17].
Broth media were used as seed culture (10% of the to-
tal volume of the fermentation medium). The culture was
adjusted to pH 7.0.
2.4. Optimization of Bacteriocins Activity
2.4.1. Production of Crude Bacteriocin Samples
Lactobacillus species were cultured in 1000 ml MRS
broth (pH 7.0) for 48 h at 30˚C. For extraction of bacte-
riocins, a cell-free solution was obtained by centrifuging
(6000 rpm for 20 min. at 4˚C) the culture and was ad-
justed to pH 7.0 [20,21].
2.4.2. Effect of Temperature on Crude Bacteriocins
Activity
In order to test the heat resistance, 10 ml of bacteriocin
preparation was heated for 30 minutes at 30˚C, 60˚C,
90˚C and 121˚C respectively. Residual bacteriocin activ-
ity was detected against E. coli and Bacillus subtilus at
each of these temperatures [22] by teterazoluim chloride
method.
2.4.3. Effect of pH on Crude Bacteriocins Activity
According to the method described by Karaoglu et al. [8],
sensitivity of the cell-free supernatant to different pH
values was tested by adjusting the pH of the bacteriocins
in the range of pH 2 to 10 with sterile IN Noah and IN
HC1. Residual activity of each of the samples was de-
termined against the indicator organism by agar-well
diffusion assay.
2.4.4. Effect of Surfactants on Crude Bacteriocins
Activity
The effect of surfactants on the bacteriocins was tested
by adding SDS, CTAB, EDTA and Tween 80 (0.5% v/v
final concentration), to crude bacteriocins. Untreated bac-
teriocin preparation (positive control). All samples were
incubated at room temperature for 2 hours then tested for
residual antimicrobial activity by teterazolium formazan
test.
2.4.5. Effect of Organic Solvents on Crude
Bacteriocins Activity
Crude bacteriocin preparations were mixed with organic
solvents including acetone, butanol, chloroform, ethanol,
methanol and propanol at a final concentration of 0.5%.
Untreated bacteriocins preparation were used as (positive
control). All samples were incubated at room tempera-
ture for 2 hours and tested for residual antimicrobial ac-
tivity by teterazolium formazan test.
2.4.6. Effect of Metal Ions on Crude Bacteriocins
Activity
In a separate experiment the effect of metal salts on bac-
teriocin was examined by adding AgNO3, CuSO4, FeSO,
MgSO4, MnCl2, and ZnSO4 (Merck) to 10 ml of crude
bacteriocin preparation (0.5% final concentration). Un-
treated bacteriocin preparation (positive control). All
samples were incubated at room temperature for 2 hours
and tested for residual antimicrobial activity [23,24] by
teterazolium formazan test.
2.4.7. Effect of Different Concentration of NaCl on
Crude Bacteriocins Activity
In a separate experiment the effect of different concen-
tration of NaCl (2%, 4%, 6%, 8%, 10%) on bacteriocins
were examined by adding to 10 ml of crude bacteriocins
preparation. Untreated bacteriocin preparation (positive
control). All samples were incubated at room tempera-
ture for 2 hours and tested for residual antimicrobial ac-
tivity [24] by agar-well diffusion assay.
2.4.8. Effect of Different Concentration of Amino
Acids on Crude Bacteriocins Activity
In a separate experiment the effect of different concen-
tration of 21 amino acids compound (essential amino
acids) (2%, 4%, 6%, 8% and 10%) on bacteriocins were
examined by adding to 10 ml of crude bacteriocins prepa-
ration. Untreated bacteriocin preparation (positive con-
trol). All samples were incubated at room temperature for
2 hours and tested for residual antimicrobial activity by
agar-well diffusion assay.
2.4.9. Effect of Different Concentration of Vitamins
on Crude Bacteriocins Activity
In a separate experiment the effect of different concen-
tration of vitamins (50%) such as (B12 and B complex)
on bacteriocins were examined by adding (1) to 10 ml of
crude bacteriocins preparation. Untreated bacteriocin
preparation (positive control). All samples were incu-
bated at room temperature for 2 hours and tested for re-
sidual antimicrobial activity by agar-well diffusion assay.
2.5. Statistical Analysis
Data are presented as the mean ± standard deviation, and
n represents the number of the isolates and the control.
3. Results and Discussion
3.1. Isolation and Identification of
Bacteriocinogenic Strains
Sixteen isolates of LAB were isolated from the samples.

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
Isolated from Some Dairy Products under Different Culture Conditions
345
After series of purification on MRS agar, four isolates
were found to be Gram-positive, catalase negative, non-
motile bacilli. In addition, all strains were tested for
growth at 10˚C for 10 days, 45˚C for 48 h, and CO2 pro-
duction from glucose [25]. Table 1 presents the results of
the final identifications for each type of isolates with API
gallery as stated below: L. acidophilus M2, L. acidophi-
lus CH1, L. fermentum M1 and L. pentosus CH2.
3.2. Bacteriocin Production
The antibacterial activity of bacteriocins against food
borne pathogenic, as well as spoilage bacteria has raised
considerable interest for their application in food preser-
vation [26]. Application of bacteriocins may help reduce
the use of chemical preservatives and/or the intensity of
heat and other physical treatments, satisfying the de-
mands of consumers for foods that are fresh tasting,
ready to eat, and lightly preserved. In the present study
the average diameter of the inhibition zones measured
ranged from 2 - 20 mm in size (Table 2). Among the
isolates, L. fermentum M1 and L. a cidophilus CH1 were
bacteriocin effectively inhibited the Bacillus subtilis
NCIB3610 with maximum inhibitory activity, compared
to the other tested bacteria while, the impact of these
strains were less against E. coli ATCC 25922. In addition,
L. a cidophilus M2 exhibited stronger inhibition activity
on Bacillus subtilis NCIB3610 than E. coli ATCC 25922
but its effect was less than the effect of L. acidophilus
CH1 that exemplifies a difference within the same spe-
cies. The present L. pentosus CH2 isolate showed inhibi-
tory activity against Ba cillus subtilis NCIB3610 on the
other hand it was less inhibitory activity against E. coli
ATCC 25922. Such observations [27,28] made earlier are
in tune with the results of the present study which con-
firmed that the bacteriocins of Gram-positive bacteria
generally exhibit antagonistic activity against Gram-posi-
tive bacteria and the activity against Gram negative bac-
teria is an unusual phenomenon and has been reported for
the bacteriocins produced by Lactobacillus plantarum
[29], the isolates were screened for antimicrobial spec-
trum against Gram-positive and Gram-negative bacteria
using the AWD method.
3.3. Effect of Culture Conditions and Medium
Composition on Bacteriocin Production
The culture conditions and composition of the growth
medium are very important for the production of indi-
vidual bacteriocins [5]. Several media have been evalu-
ated by numerous authors to improve bacteriocin synthe-
sis [30] because these peptides are not always produced
in standard or enriched culture media. Lactic acid bacte-
ria are fastidious microorganisms that require rich media
containing milk, whey ultrafiltrate, or complex synthetic
media such as MRS [10], M17 [31] or LAPTg [32] for
growth. Therefore, the isolation of a peptide(s) in rich-
medium supernatant is an additional problem, making the
purification of the bacteriocin a relatively complicated
protocol. The present study was primarily aimed to de-
termine cultural conditions for obtaining better and stable
bacteriocins production. L. acidophilus CH1 was able to
produce bacteriocins, which had a wide inhibitory spec-
trum towards both Gram-negative and Gram-positive food
spoilage and pathogenic bacteria. Results show that bac-
teriocin was produced when nutrients were available for
metabolic activity. Tables 3 and 4 showed that maximum
activity was noted at pH 6.0, temperature 30˚C. Bacte-
riocin production is frequently regulated by pH and
growth temperature, as has been shown in several studies
involving the pediocin AcH [33]. From the results proved
that it could be used in acidic foods like pickle or yoghurt.
It might be secondary metabolites.
The composition of medium influencing the produc-
tion of bacteriocin by Lactobacillus isolates. Table 5
showed that MRS seemed to be more suitable medium
Table 1. Pre-identification of some isolates.
Preliminary tests
Strain Gram staining Catalase test Growth at 10˚C Growth at 45˚C CO2 Production API Identification
M2 + − − + − L. acidophilus
CH1 + − − + − L. acidophilus
CH2 + − − + + L. pentousus
M1 + − + + − L. fermentum
Table 2. Bacteriocin production by four isolates Detected by well-diffusion assay.
Strains Diameter of the inhibition-zone (mm) for B. subtilis Diameter of the inhibition-zone (mm) for E. coli
L. fermentum M1 20 ± 0.01 3 ± 0.02
L. acidophilus M2 18 ± 0.02 16 ± 0.03
L. acidophilus CH1 20 ± 0.01 18 ± 0.01
L. pentousus CH2 15 ± 0.01 2 ± 0.02
Data are presented as mean ± SD.

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
Isolated from Some Dairy Products under Different Culture Conditions
346
Table 3. Effect of different temperature on production.
Diameter of the inhibition-zone (mm)
for E. coli ATCC 25922
Diameter of the inhibition-zone (mm)
for Bacillus subtilis NCIB3610
Temp.
Strains L. fermentum
M1
L. acidophilus
M2
L. acidophilus
CH1
L. pentosus
CH2
L. fermentum
M1
L. acidophilus
M2
L. acidophilus
CH1
L. pentosus
CH2
Control 20 ± 0.01 3 ± 0.01 9 ± 0.01 5 ± 0.01 3 ± 0.01 4 ± 0.01 16 ± 0.01 2 ± 0.01
10 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
20 3 ± 0.01 4 ± 0.01 6 ± 0.01 4 ± 0.01 5 ± 0.01 4 ± 0.01 14 ± 0.01 6 ± 0.01
30 4 ± 0.01 3 ± 0.01 16 ± 0.01 7 ± 0.01 4 ± 0.01 5 ± 0.01 21 ± 0.01 7 ± 0.01
40 3 ± 0.01 5 ± 0.01 9 ± 0.01 3 ± 0.01 3 ± 0.01 2 ± 0.01 15 ± 0.01 6 ± 0.01
50 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Data are presented as mean ± SD.
Table 4. Effect of different pH on production.
Diameter of the inhibition-zone (mm)
for E. coli ATCC 25922
Diameter of the inhibition-zone (mm)
for mBacillus subtilis NCIB3610
pH
Strains L. fermentum
M1
L. acidophilus
M2
L. acidophilus
CH1
L. pentosus
CH2
L. fermentum
M1
L. acidophilus
M2
L. acidophilus
CH1
L. pentosus
CH2
Control 2 ± 0.01 3 ± 0.01 9 ± 0.01 5 ± 0.01 3 ± 0.01 4 ± 0.01 16 ± 0.01 2 ± 0.01
2 1 ± 0.01 0.0 2 ± 0.01 0.0 0.0
1 ± 0.01 3 ± 0.01 0.0
4 2 ± 0.01 1 ± 0.01 3 ± 0.01 1 ± 0.01 0.0 1 ± 0.01 4 ± 0.01 0.0
6 2 ± 0.01 3 ± 0.01 10 ± 0.01 7 ± 0.01 4 ± 0.01 4 ± 0.01 20 ± 0.01 7 ± 0.01
8 1 ± 0.01 2 ± 0.01 5 ± 0.01 2 ± 0.01 2 ± 0.01 2 ± 0.01 10 ± 0.01 10 ± 0.01
10 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Data are presented as mean ± SD.
for the bacteriocin production. Similar results were ob-
served by [34,35]. Results in Table 5 also indicate that
larger amounts of the bacteriocins were synthesized only
in MRS medium supplemented with K2HPO4 (1.0%),
Tween 80 (1%), Beef extract (1%), glucose, cyctein and
peptone extract (1%), while addition of tri-ammonium
citrate, sodium acetate and magnesium sulphate, had no
effect on bacteriocin production. Thus variation in the
concentration of constituents/ supplementation of culti-
vation media might have an influence on the amount of
bacteriocin produced by microorganisms. Similar obser-
vations have been made previously. Daba et al. [21] ob-
tained similar results in the production of mensenterocin
5. Biswas et al. [33] compared the production of pediocin
ACH by Pediococcus acidilactici H cultivated in TGE
broth, MRS broth and several modifications of it. Modi-
fication of nutrients of cultivation media should be con-
sidered for maximal production of bacteriocin that has
potential use as a food biopreservative [33]. Similar re-
sults were recorded for nisin [36] and pediocin AcH [33].
The reason for increased bacteriocin production is not
clear and yet to be ascertained. Most of the bacteriocin
producing organisms requires stabilizers or a unique me-
dium composition for bacteriocin synthesis. It is probable
that the yeast extract may in part serve to inactivate an
inhibitor of bacteriocin synthesis [37]. Being a surfactant
Tween 80, might enable the discharging of the bacterio-
cin from the cell surface of the producer strain. This
finding was supported by the increased bacteriocin pro-
duction in the medium supplemented with different con-
centrations of yeast extract plus Tween-80.
An earlier study by the senior author [28] revealed that
in L. plantaram MTCC1746, maximum bacteriocin pro-
duction could be achieved by providing 1.5% yeast ex-
tract and 1.5% Tween-80. The addition of MgSO4 could
make a slight impact on the production of bacteriocin.
Activity of 1000 AU/mL was observed by the addition of
this substrate at a lower concentration of 0.02% to 0.04%.
The higher concentrations 1%, 2% and 3%, however,
bring about reduction in bacteriocin production.
3.4. Influence of Different Media on the
Production of Bacteriocins
Several complex culture media of high cost have been
used for bactenocins production. In the current study, we
have used an effluent from the food industry (Corn sleep

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
Isolated from Some Dairy Products under Different Culture Conditions 347
Table 5. Effect of adding some nutrient components in MRS media on bacteriocins production by Lactobacillus spp. isolates.
Diameter of the inhibition-zone (mm)
Medium Constituents % E. coli ATCC 25922 Bacillus subtilis NCIB3610
M1 M2 CH1 CH2 M1 M2 CH1 CH2
MRS (control) 2 3 9 5 3 4 16 2
1 0.00 3 9 0.00 0.00 0.00 10 0.00
2 0.00 0.00 0.00 0.00 0.00 0.00 9 0.00 MRS + Yeast extract
3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
1 10 0.00 13 0.00 0.00 0.00 25 0.00
2 15 0.00 17 10 0.00 0.00 0.00 0.00 MRS + Beef extract
3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
1 0.00 0.00 0.00 0.00 5 7 24 0.00
2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
MRS + Peptone extract
3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
1 0.00 0.00 0.00 0.00 0.00 0.00 13 0.00
2 0.00 0.00 15 0.00 0.00 0.00 11 0.00 MRS + Glucose
3 9 19 11 9 0.00 0.00 0.0 0.00
1 0.00 0.00 12 0.00 0.00 0.00 23 0.00
2 0.00 0.00 0.00 0.00 0.00 0.00 15 0.00 MRS + Tween80
3 0.00 0.00 5 9 8 12 20 0.00
1 1 2 3 5 9 0.00 19 0.00
2 2 2 4 2 0.00 0.00 0.00 0.00 MRS + Sodium acetate
3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
1 4 0.00 0.00 0.00 0.00 5 10 0.00
2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 MRS + Tri-ammonium citrate
3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
1 0.00 11 12 5 6 6 16 0.00
2 0.00 5 0.0 0.00 7 0.0 0.0 0.00
MRS + MgSo4·7H2O
3 0.00 0.0 0.0 0.0 0.0 0.0 0.0 0.00
1 12 10 20 0.00 0.00 0.00 15 0.00
2 0.00 0.00 15 0.00 0.00 0.00 11 0.00 MRS + Cyctein
3 0.00 0.00 25 15 0.00 0.00 10 0.00
1 0.00 0.00 0.00 10 9 20 27 0.00
2 0.00 0.00 0.00 9 0.00 9 13 0.00 MRS + K2HPO4
3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
liquar (CSL), CSL with glucose and glysrol culture me-
dia for bacteriocins production of at low costs. These
media were used for bactenocin production by four lactic
acid bacteria isolates (Lactobacillus fermentum M1,
Lactobacillus acidophilus M2, Lactobacillu s acidophilus
CH1 and Lactobacillus pentosus CH2). Production of
bacteriocins at 30˚C and at a pH 6.5 were carried out in
different media MRS, Medium (A) Corn steep liquor—
Lactose medium, Medium (B) Corn steep liquor—Lac-
tose medium, Medium (C) Corn steep liquor medium,
Medium (D) Glycerol-molasses-liquid medium for bac-
teriocins (Table 6). A maximum growth rate were shown
in MRS and CSL for all isolates and a maximum bacte-
riocrn activity (inhibition zone mm of Bacillus subtilis
NCIB3610) was appeared by-isolate Lactoba cillus aci-
dophilus CH1 in medium C, but the maximum bacte-
riocm activity by isolate CH1. On the other hand isolates
CHl and M1 were given the maximum bacteriocm(s)
activity in meadium C. The lowest amounts of bacterio-
cins activity were produced in date by M1 isolate. Al-
though this fact suggests the possible effect of substrate
inhibition, it could also be related to the control that the
supplied sugar substrate exerts on the bacteriocm bio-
synthesis. Biswas et al. [33] reported that MRS medium
is a better medium for cell growth and bacteriocins pro-
duction than other media. Generally, maximum produc-
tion corresponds to against pathogenic microbe such as
Bacillus subtilis NCIB3610 and E. coli ATCC 25922.

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
Isolated from Some Dairy Products under Different Culture Conditions
348
Table 6. Effect of different media on production of bacteriocins.
Strains Diameter of the inhibition-zone (mm)
for Bacillus subtilis NCIB3610
Diameter of the inhibition-zone (mm)
for E. coli ATCC 25922
Media L. fermentum
M1 L. acidophilus M2 L. acidophilus CH1 L. pentosus CH2 L. fermentum M1 L. acidophilum M2 L. acidophilus CH1 L. pentosus CH2
MRS control 2 3 9 5 3 4 16 2
A 8 0.0 10 0.0 0.0 0.0 0.0 0.0
B 8 5 20 12 0.0 0.0 0.0 0.0
C 5 25 32 20 0.0 0.0 0.0 0.0
D 10 0.0 16 0.0 0.0 0.0 0.0 0.0
Medium (A) Corn steep liquor-Lactose medium. Medium (B) Corn steep liquor-Lactose medium with some modifications. Medium (C) Corn
steep liquor medium. Medium (D) Glycerol-molasses-liquid medium.
Therefore increased cell concentrations in a high cell-
density reactor is expected to increase bactenocm pro-
duction. In general bactencin production by lactic acid
bacteria occurs during the active growth phase [29,38].
Conditions favouring bacterial growth and high cell den-
sities are frequently beneficial to bactenocin production
as well [38]. However, a high cell yield does not neces-
sarily result in a high bactenocin activity since the latter
may be limited by a low specific bacteriocin production,
i.e. a low bacteriocin production per gram of cells [28].
Hence, there exists a rather complex relationship be-
tween environmental conditions and bactenocin activity
levels and no generalisation about the optimum condi-
tions for bactenocin production can readily be made. The
kinetics of both cell growth and bactenocin production in
function of the environmental situation have to be stud-
ied to obtain a better understanding of the production
mechanism.
3.5. Optimization of Bacteriocin Activity
3.5.1. Effect of Different Temperatures on the Crud
Bacteriocin
The effect of different temperatures on crud bacteriocin
have been clarified in the Tables 7-10. These tables
clearly highlights of effect of different temperature 30˚C,
60˚C and 90˚C /30min on crud bacteriocin from four
LAB: L. fermentum M1, L. acidoph ilus M2, L. acidophi-
lus CH1and L. pentosus CH2 according to the tetera-
zolium chloride methods. As can be seen, LAB were
isolated from local raw milk and Ras cheese using MRS
agar. According to Table 7, the isolated Lactobacillus
fermentum M1 was showed antimicrobial activity against
E.coli ATCC 25922 which showed the largest of growth
inhibitor% around 76.08% in temp. 60˚C/30min bacte-
riocin but the smallest of the antimicrobial activity was
39.34% in temp. 90˚C/30min wherever Lactobacillus
fermentum was showed antimicrobial activity against Ba-
cillus subtilis NCIB3610 was 35.79% in temp. 60˚C/30min
but the smallest of the antimicrobial activity was 30% in
temp. 30˚C/30min. Table 8, in addition, the strains L.
acidophilus M2 which showed the largest growth inhibi-
tion% was 80.32% in temp. 60˚C/30min against Bacillus
subtilis NCIB3610 but the smallest was 65.81% in temp.
90˚C/30min wherever, the same strain was the largest
growth inhibitor% 87.34% in temp. 60˚C/30min but the
smallest was 11.12% in temp. 30˚C/30min against to E.
coli.
According to Table 9 The isolated Lactobacillus aci-
dophilus CH1 was showed antimicrobial activity against
E.coli ATCC 25922 which showed the largest of growth
inhibitor% around 59.56% in temp. 60˚C/30min but that
showed the smallest 18.08% in temp. 90˚C/30min. Bac-
teriocin wherever La ctobacillus acidophilus CH1 was
showed antimicrobial activity against Bacillus subtilis
NCIB3610 was 87.08% in temp. 30˚C/30min but the
smallest of the antimicrobial activity was 18.08% in temp.
90˚C/30min. Table 10, in addition, the strains L. pento-
sus CH2 which showed the largest growth inhibition%
was 85.98% in temp. 60˚C/30min against Bacillus sub-
tilis NCIB3610 but the smallest was 0.00% in temp.
60˚C/30min wherever, the same strain was the largest
growth inhibitor% 98.40% in temp. 90˚C/30min against
to E. coli.
An optimal temperature of 25˚C for the production of
bacteriocin by Leuconostoc carnosum LA54A was found
by Geisen et al. [39]. Vignolo et al. [40] showed that
production of lactocin 705 by Lactobacillus casei CRL
705 increased as the culture temperature was reduced: for
every temperature tested (15˚C ± 30˚C), bacteriocin pro-
duction levels were identical but biomass increased with
the temperature. Moreover, the lower the temperature,
the higher the volumic production, particularly for mes-
enterocin 52 A. This indicated that bacteriocin produc-
tion was stimulated by temperatures unfavorable for
growth, particularly the low temperatures.
3.5.2. Effect of Different Levels of pH on the Crud
Bacteriocin
The effects of different pH such as 2, 4, 6, 8 and 10 on
crude of bacteriocins were studied. In MRS broth, pH2

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
Isolated from Some Dairy Products under Different Culture Conditions 349
Table 7. Effect of temperature on crude bacteriocins.
L. fermentum M1
Bacillus subtilis NCIB3610 E. coli ATCC 25922
O.D Growth % Growth inhibitor % O.D Growth % Growth inhibitor %
Control 0.813 100 0.00 1.350 100 0.00
30˚C/30min 0.569 69.99 30.01 0.531 39.33 60.67
60˚C/30min 0.522 64.21 35.79 0.323 23.93 76.08
90˚C/30min 0. 55 67.65 32.34 0.090 6.66 39.34
Table 8. Effect of temperature on crude bacteriocins.
L. acidophilus M2
Bacillus subtilis NCIB3610 E. coli ATCC 25922
O.D Growth % Growth inhibitor % O.D Growth % Growth inhibitor %
Control 0.813 100 0.00 1.350 100 0.00
30˚C/30min 0.232 28.54 71.46 0.943 69.85 30.15
60˚C/30min 0.160 19.68 80.32 0.171 12.66 87.34
90˚C/30min 0.278 34.19 65.81 1.20 88.88 11.12
Table 9. Effect of temperature on crude bacteriocins.
L. acidophilus CH1
Bacillus subtilis NCIB3610 E. coli ATCC 25922
O.D Growth % Growth inhibitor % O.D Growth % Growth inhibitor %
Control 0.813 100 0.00 1.350 100 0.00
30˚C/30min 0.105 12.92 87.08 1.007 74.59 25.41
60˚C/30min 0.411 50.55 49.45 0.546 40.44 59.56
90˚C/30min 0.666 81.92 18.08 1.096 81.19 18.82
Table 10. Effect of temperature on crude bacteriocin.
L. pentosus CH2
Bacillus subtilis NCIB3610 E. coli ATCC 25922
O.D Growth % Growth inhibitor % O.D Growth % Growth inhibitor %
Control 0.813 100 0.00 1.350 100 0.00
30˚C/30min 0.122 15 84.99 0.221 16.37 83.63
60˚C/30min 0.114 14.02 85.98 0.221 16.37 83.63
90˚C/30min 1.229 151.17 −51.17 2.154 1.59 98.40
increased the activity of bacteriocins isolated from Lac-
tobacillus acidophillu s CH1 against to E. coli ATCC
25922 was 12 mm but the L. acidophillus M2, L. fer-
mentum M1 and L. pentosus CH2 isolates were around (5,
9, 10) mm, wherever four isolates were decreased pH
against to Bacillus subtilis NCIB3610 (Table 11). Among
them, in pH 6, the largest activity of bacteriocin from
Lactobacillus acidophilus CH1, L. acidophillus M2, L.
fermentum M1 and L. pentosus CH2 against E. coli ATCC
25922 was shown 11, 11, 28 and 15 mm, but isolates
were decreased pH against to Bacillus subtilis NCIB3610.
Among them, in pH 8, the largest activity of bacteriocin
from Lactobacillus acidophilus CH1, L. acidophillus M2,
L. fermentum M1 and L. pentosus CH2 against E. coli
ATCC 25922 was shown 13, 10, 26 and 20 mm, but iso-
lates were decreased pH against to Bacillus subtilis
NCIB3610. In pH 10, no activity for bacteriocin from
Lactobacillus spp. against E. coli ATCC 25922 and Ba-
cillus subtilis NCIB3610. So that, the activity of different
bacteriocins were shown in pH6 against E. coli ATCC
25922 and the isolates were decreased against to Bacillus
subtilis NCIB3610.
Thus, under uncontrolled pH conditions, a lower tem-
perature coincided with a higher maximum bacteriocin
production, a result also obtained by De Vugst et al. [41]
with the bacteriocin from Lactobacillus amylovorus.

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
Isolated from Some Dairy Products under Different Culture Conditions
350
Table 11. Effect of pH on crude bacteriocins.
E. coli ATCC 25922 Bacillus subtilis NCIB3610
pH
Strains
L. fermentum
M1
L. acidophilus
M2
L. acidophilus
CH1
L. pentosus
CH2
L. fermentum
M1
L. acidophilus
M2
L. acidophilus
CH1 L. pentosus CH2
2 10 9 12 5 6 8 9 5
4 9 11 9 9 10 8 10 10
6 11 11 28 20 5 5 33 5
8 13 10 26 15 5 5 9 5
10 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
3.5.3. Effect of Surfactant on Crude Bacteriocins
Tables 12-15 clearly highlights of effect of different
minerals such as SDS, ETDA, Tween80 and CTAB on
crud bacteriocin from four LAB such as L. fermentum
M1, L. acidophilus M2, L. acidophilus CH1and L. pen-
tosus CH2 according to the teterazolium chloride meth-
ods. As can be seen, LAB were isolated from local raw
milk and Ras cheese using MRS agar. According to Ta-
ble 12 The isolated Lactobacillus fermentum M1 was
showed antimicrobial activity against E. coli ATCC 25922
which showed the largest of growth inhibitor% around
73.56% in Tween80 on bacteriocin but the smallest of
the antimicrobial activity was 34.23% in CTAB wher-
ever Lactobacillus fermentum was showed antimicrobial
activity against Bacillus subtilis NCIB3610 was 47.48%
in Tween80 but the smallest of the antimicrobial activity
was 12.42% in SDS. Table 13, in addition, the strains L.
acidophilus M2 which showed the largest growth inhibi-
tion% was 78.59% in SDS against Bacillus sub tilis
NCIB3610 but the smallest was 11.44% in Tween80
wherever, the same strain was the largest growth inhibi-
tor% 74.52% in CTAB but the smallest was 39.4% in
Tween80 against to E. coli.
According to Table 14, the isolated Lactoba cillus
acidophilus CH1 was showed antimicrobial activity
against E. coli ATCC 25922 which showed the largest of
growth inhibitor% around 78.29% in Tween80 but that
showed the smallest 20% in CTAB. Bacteriocin wher-
ever Lactobacillus acidophilus CH1 was showed antim-
icrobial activity against Bacillus su btilis NCIB3610 was
75.77% in Tween80 but the smallest of the antimicrobial
activity was 34.44% in SDS. Table 15, in addition, the
strains L. pentosus CH2 which showed the largest growth
inhibition% was 49.57% in CTAB against Bacillus sub-
tilis NCIB3610 but the smallest was 7.13% in Tween80
wherever, the same strain was the largest growth inhibi-
tor% 80.59% in CTAB against to E. coli but the smallest
of the antimicrobial activity was 40.89% in ETDA. Simi-
lar observation was made earlier in L. acidophilus [42].
3.5.4. Effect of Organic Solvents on Crude
Bacteriocins Activity
Tables 16-19 clearly highlights of effect of different sol-
vent such as ethanol, Isopropanol, Isoamylchlorde and
chrolform on crud bacteriocin from four LAB such as L.
fermentum M1, L. acidophilus M2, L. acidophilus CH1and
L. pentosus CH2 according to the teterazolium chloride
methods.
As can be seen, LAB were isolated from local raw
milk and Ras cheese using MRS agar. According to Ta-
ble 16, the isolated Lactobacillus fermentum M2 was
showed antimicrobial activity against E. coli ATCC 25922
which showed the largest of growth inhibitor% around
60.67% in ethanol bacteriocin wherever Lactobacillus
fermentum was showed antimicrobial activity against
Bacillus subtilis NCIB3610 was 93.85% in Iso amyl
chloride but the smallest of the antimicrobial activity was
30% in ethanol. Table 17, in addition, the strains L. aci-
dophilus M2 which showed the largest growth inhibi-
tion% was 88.98% in chroloform against Bacillus subtilis
NCIB3610 but the smallest was 46.87% in ethanol wher-
ever, the same strain was the largest growth inhibitor%
87% in isopropanol but the smallest was 11.12% in Iso
amyl chloride against to E. coli.
According to Table 18, the isolated Lactobacillus aci-
dophilus CH1 was showed antimicrobial activity against
E. coli ATCC 25922 which showed the largest of growth
inhibitor% around 85.19% in ethanol but that showed the
smallest 38.45% in isoamylchlorde. Bacteriocin wher-
ever Lactobacillus acidophilus CH1 was showed antim-
icrobial activity against Bacillus su btilis NCIB3610 was
87.69% in ethanol but the smallest of the antimicrobial
activity was 14.69% in chloroform. Table 19, in addition,
the strains L. pentosus CH2 which showed the largest
growth inhibition% was 33.21% in ethanol against Ba -
cillus subtilis NCIB3610 but the smallest was 0.00% in
chloroform wherever, the same strain was the largest
growth inhibitor% 70.52% in ethanol but the smallest
was 40.74% in isopropanol against to E. coli.
3.5.5. Effect of Some Minerals Salt on Crude
Bacteriocins
Tables 20-23 clearly highlights of effect of different
minerals such as AgNo3, CuSo4, FeSo4, MgSo4, MnCl2
and ZnSo4 on crud bacteriocin from four LAB isolates
according to the teterazolium chloride methods. Tables

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
Isolated from Some Dairy Products under Different Culture Conditions 351
Table 12. Effect of surfactant on crude bacteriocins from Lactobacillus fermentum M1.
Lactobacillus fermentum M1
Baccillus subtilis NCIB3610 E. coli ATCC 25922
Isolates
Surfactant O.D Growth % Growth inhibitor % O.D Growth % Growth inhibitor %
Control 0.813 100 0.00 1.350 100 0.00
SDS 0.712 87.58 12.42 0.739 54.74 45.26
ETDA 0.576 71.09 28.91 1.766 130.81 −30.81
Tween80 0.427 52.52 47.48 0.357 26.44 73.56
CTAB 0.465 57.19 42.80 0.888 65.78 34.23
Table 13. Effect of surfactant on crude bacteriocins from Lactobacillus acidophillus M2.
L. acidophillus M2
Baccillus subtilis NCIB3610 E. coli ATCC 25922
Isolates
Surfactant O.D Growth % Growth inhibitor % O.D Growth % Growth inhibitor %
Control 0.813 100 0.00 1.350 100 0.00
SDS 0.174 21.40 78.59 0.549 40.67 59.34
ETDA 0.272 33.46 66.54 1.463 108.37 −8.37
Tween80 0.720 88.56 11.44 0.818 60.59 39.4
CTAB 0.583 71.71 28.29 0.344 25.48 74.52
Table 14. Effect of surfactant on crude bacteriocins from Lactobacillus acidophillus CH1.
L. acidophillus CH1
Baccillus subtilis NCIB3610 E. coli ATCC 25922
Isolates
Surfactant O.D Growth % Growth inhibitor % O.D Growth % Growth inhibitor %
Control 0.813 100 0.00 1.350 100 0.00
SDS 0.533 65.56 34.44 0.359 26.59 73.41
ETDA 0.494 60.76 39.24 1.670 123.70 −23.70
Tween80 0.197 24.23 75.77 0.293 21.70 78.29
CTAB 0.292 35.92 64.08 1.080 80 20
Table 15. Effect of surfactant on crude bacteriocins from Lactobacillus pentosus CH2.
L. pentosus CH2
Baccillus subtilis NCIB3610 E. coli ATCC 25922
Isolates
Surfactant O.D Growth % Growth inhibitor % O.D Growth % Growth inhibitor %
Control 0.813 100 0.00 1.350 100 0.00
SDS 0.512 62.98 37.02 1.907 141.25 −14.25
ETDA 0.503 61.87 38.13 0.798 59.11 40.89
Tween80 0.755 92.87 7.13 2.499 185.11 −85.11
CTAB 0.410 50.43 49.57 0.262 19.41 80.59
Table 16. Effect of different solvent on crude bacteriocin.
Lactobacillus fermentum M1
Bacillus subtilis NCIB3610 E. coli ATCC 25922
Different Solvents
O.D Growth % Growth inhibitor % O.D Growth % Growth inhibitor %
Control 0.813 100 0.00 1.350 100 0.00
Ethanol 0.569 69.99 30.01 0.531 39.33 60.67
Isopropanol 0.522 64.21 35.79 1.323 98 2
Iso amyl chlode 0.05 6.15 93.85 0.90 66.66 33.34
Chroloform 0.554 68.14 31.86 0.00 0.00 0.00

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
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352
Table 17. Effect of different solvent on crude bacteriocin.
Lactobacillus acidophilus M2
Bacillus subtilis NCIB3610 E. coli ATCC 25922
Different Solvents
O.D Growth % Growth inhibitor %O.D Growth % Growth inhibitor %
Control 0.813 100 0.00 1.350 100 0.00
Ethanol 0.10 12.30 87.69 0.20 14.81 85.19
Isopropanol 0.683 84.00 15.99 0.767 56.81 43.19
Iso amyl chloride 1. 90 233.7 −33.7 0.831 61.56 38.45
Chroloform 0.692 85.12 14.89 0.739 54.74 45.26
Table 18. Effect of different solvent on crude bacteriocin.
Lactobacillus acidophilus CH1
Bacillus subtilis NCIB3610 E. coli ATCC 25922 Different Solvents
O.D Growth % Growth inhibitor %O.D Growth % Growth inhibitor %
Control 0.813 100 0.00 1.350 100 0.00
Ethanol 0.432 53.14 46.87 0.943 69.85 30.15
Isopropanol 0.10 12.30 87.69 0.171 12.66 87.34
Iso amyl chlode 0.278 34.19 65.81 1.20 88.88 11.12
Chroloform 0.090 11.07 88.93 2.00 148.15 −48.15
Table 19. Effect of different solvent on crude bacteriocin.
Lactobacills pentosus CH2
Bacillus subtilis NCIB3610 E. coli ATCC 25922
Different Solvents
O.D Growth % Growth inhibitor %O.D Growth % Growth inhibitor %
Control 0.813 100 0.00 1.350 100 0.00
Ethanol 0.543 66.79 33.21 0.398 29.48 70.52
Isopropanol 0.765 94.09 5.91 0.800 59.26 40.74
Iso amyl chlode 0.625 76.88 23.12 0.699 51.78 48.23
Chroloform 0.00 0.00 0.00 0.421 30.52 69.48
showed that LAB were isolated from local raw milk and
Ras cheese using MRS agar. According to Table 20 The
isolated Lactobacillus fermentum M1 was showed an-
timicrobial activity against E. coli ATCC 25922 which
showed the largest of growth inhibitor% around 86.89%
in MgSo4 on bacteriocin but the smallest of the antim-
icrobial activity was 12.23% in FeSo4 wherever Lacto-
bacillus fermentum was showed antimicrobial activity
against Ba cillus subtilis NCIB3610 was 87.20% in FeSo4
but the smallest of the antimicrobial activity was 39.36%
in CuSo4. Table 21 showed that the L. acidophilu s M2
isolate which showed the largest growth inhibition% was
95.57% in FeSo4 against Bacillus subtilis NCIB3610 but
the smallest was 46.74% in CuSo4 wherever, the same
strain was the largest growth inhibitor% 77.70% in
MgSo4 but the smallest was 40.37% in FeSo4 against to E.
coli.
According to Table 22, the Lactobacillus acidophilus
CH1 isolate was showed antimicrobial activity against
E.coli ATCC 25922 which showed the largest of growth
inhibitor% around 97.92% in FeSo4 but that showed the
smallest 66.89% in MgSo4. Bacteriocin wherever Lacto-
bacillus acidophilus CH1 was showed antimicrobial ac-
tivity against Bacillus su btilis NCIB3610 was 75.03% in
ZnSo4 but the smallest of the antimicrobial activity was
5.41% in CuSo4. Table 23, in addition, the strains L.
pentosus CH2 which showed the largest growth inhibi-
tion% was 97.17% in FeSo4 against Bacillus subtilis
NCIB3610 but the smallest was 15.5% in ZnSo4 wher-
ever, the same strain was the largest growth inhibitor%
93.11% in MgSo4 against to E. co li but the smallest of
the antimicrobial activity was 13.34% in AgSo4.
3.5.6. Effect of Different Concentration of NaCl on
Crude of Bacteriocins
The effects of different concentration of NaCl on crude
of bacteriocins were studied. In MRS broth, 2% NaCl
increased the activity of bacteriocins isolated from Lac-
tobacillus pentosus CH2 against to E.coli ATCC 25922
was 14 mm, L. fermen tum M1 was shown15 mm against
to Bacillus subtilis NCIB3610 (Table 24). Among them,
bacteriocin from Lactobacillus pentosus CH2 against

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
Isolated from Some Dairy Products under Different Culture Conditions 353
Table 20. Effect of some mineral salt on crude bacteriocins from Lactobacillus fermentum M1.
Lactobacillus fermentus M1
Bacillus subtilis NCIB3610 E. coli ATCC 25922
O.D Growth % Growth inhibitor %O.D Growth % Growth inhibitor %
control 0.813 100 0.00 1.350 100 0.00
AgNO3 0.844 103.8 −3.8 2.221 164.5 −64.5
CuSO4 0.493 60.63 39.36 2.221 164.5 −64.5
FeSO4 0.104 12.79 87.20 1.185 87.76 12.23
MgSO4 0.301 37.03 62.98 0.177 13.11 86.89
MnCl2 0.216 26.57 73.43 2.301 170.44 −70.44
ZnSO4 0.424 52.15 47.85 0.975 72.22 27.77
Table 21. Effect of some mineral salt on crude bacteriocins from Lactobacillus acidophillus M2.
Lactobacillus acidophillus M2
Bacillus subtilis NCIB3610 E. coli ATCC 25922
O.D Growth % Growth inhibitor %O.D Growth % Growth inhibitor %
control 0.813 100 0.00 1.350 100 0.00
AgNO3 0.448 55.10 44.89 1.305 96.67 3.34
CuSO4 0.433 53.26 46.74 1.793 132.8 −32.8
FeSO4 0.036 4.43 95.57 0.805 59.63 40.37
MgSO4 0.09 11.07 88.92 0.301 22.29 77.70
MnCl2 0.833 102.46 −2.46 2.040 151.11 −51.11
ZnSO4 0.297 36.53 63.47 2.20 162.96 −62.96
Table 22. Effect of some mineral salt on crude bacteriocins from Lactobacillus acidophillus CH1.
Lactobacillus acidophillus CH1
Bacillus subtilis NCIB3610 E. coli ATCC 25922
O.D Growth % Growth inhibitor %O.D Growth % Growth inhibitor %
control 0.813 100 0.00 1.350 100 0.00
AgNO3 0.665 81.79 18.20 2.096 155.25 −55.25
CuSO4 0.769 94.59 5.41 2.301 170.44 −70.44
FeSO4 0.265 32.59 67.40 0.028 2.07 97.92
MgSO4 0.301 37.02 62.97 0.447 33.11 66.89
MnCl2 0.700 86.10 13.89 2.484 184 −84
ZnSO4 0.203 24.97 75.03 2.444 181.03 −81.03
Table 23. Effect of some mineral salt on crude bacteriocins from Lactobacillus pentosus CH2.
Lactobacillus pentosus CH2
Bacillus subtilis NCIB3610 E. coli ATCC 25922
O.D Growth % Growth inhibitor %O.D Growth % Growth inhibitor %
control 0.813 100 0.00 1.350 100 0.00
AgNO3 0.543 66.79 33.21 1.170 86.67 13.34
CuSO4 0.685 84.26 15.74 2.461 182.3 −82.3
FeSO4 0.023 2.83 97.17 0.334 24.74 75.26
MgSO4 0.201 24.73 75.27 0.093 6.89 93.11
MnCl2 0.687 84.50 15.5 2.470 182.9 −82.96
ZnSO4 0.230 28.3 71.7 0.155 11.48 88.51

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
Isolated from Some Dairy Products under Different Culture Conditions
354
E. coli ATCC 25922 was shown 12 mm, L. fermentum
M1 was shown13 mm against to Bacillus subtilis
NCIB3610 was showed activity in the presence of 4%
NaCl concentration, but this activity at 6% and 8% NaCl.
Two bacteriocins from L. pentosus CH2 and L. aci-
dophilus M2 sawed 11 or 10 mm. According to 10%
NaCl concentration were shown no increase in their ac-
tivity, but were inhibited by more than 1% NaCl in MRS
media. The supplementation with NaCl, bacteriological
peptone and beef extract have resulted in reduced activity.
In contrast to the present observation, growth as well as
bacteriocin production in the presence of bacteriological
peptone or casamino acids and NaCl was reported to be
higher by previous researchers [43,44].
3.5.7. Effect of Different Concentration of Amino
Acids Component on Crude Bacteriocin
Activity
The effects of different concentrations of 21 esintial
amino acid such as 2%, 4%, 6%, 8% and 10% on crude
of bacteriocins were studied Table 25. In MRS broth, 2%
increased the activity of bacteriocins isolated from Lac-
tobacillus pentosus CH2 against to E. coli ATCC 25922
was 9 mm but the L. fermentum M1 was around (8 mm)
activity, the L. acidophillus M2 and L. pentosus CH2
were no activity. The activity of L. fermentum M1 was
showed 13 mm against to Bacillus subtilis NCIB3610.
Among them, in 4%, the largest activity of bacteriocin
from Lactobacillus pentosus CH2 against E. coli ATCC
25922 was shown 10 mm, but isolates were no activity
against to Bacillus subtilis NCIB3610. Among them, 6%,
the largest activity of bacteriocin from L. acidophillus
M2 against E. coli ATCC 25922 was shown 11 mm, but
isolates were no activity against to Bacillus subtilis
NCIB3610. 8%, the largest activity of bacteriocin from L.
fermentum M1 against E. co li ATCC 25922 was shown
11 mm, but isolates were no activity against to Bacillus
subtilis NCIB3610. In 10%, no activity for bacteriocin
from Lactobacillus sp. against E. coli ATCC 25922 and
Bacillus subtilis NCIB3610. So that, the activity of dif-
ferent bacteriocins were shown in 2% against E. coli
ATCC 25922 and Bacillu s subtilis NCIB3610.
3.5.8. Effect of Different Vitamins Component on
Crude Bacteriocin Activity
The effects of different vitamins such as B12 and B com-
plex on crude of bacteriocins were studied Table 26. In
MRS broth, B12 increased the activity of bacteriocins
isolated from Lactobacillus acidoph illus CH1 against to
E. coli ATCC 25922 was 10 mm but the three other iso-
lates no activity were observed, wherever four isolates
were shown 10 to 11 mm against to Bacillus subtilis
NCIB3610. Among them, in B complex, bacteriocin
from Lactobacillus acidophilus CH1 against E. coli ATCC
25922 was shown 13 to 14 mm, the largest activity of
bacteriocins for L. fermentum M1 and L. acidophilus
CH1were shown15 mm against to Bacillus subtilis
NCIB3610 and the smallest was showed 5 mm in L. aci-
dophilus M2 and L. pentosus CH2. This result was agree
with Adenike et al. [45].
Table 24. Effect of different concentration of Nacl on crude bacteriocin.
E. coli ATCC 25922 Bacillus subtilis NCIB3610
Na Cl
Strains
L.
fermentum M1 L. acidophils M2 L. Acidophils CH1
L.
pentosus CH2
L.
Fermentum M1
L.
Acidophils M2 L. Acidophils CH1 L. pentosus CH2
Control 2 3 9 5 3 4 16 2
2% 10 11 11 14 15 14 10 10
4% 9 6 10 12 13 11 9 11
6% 0.0 0.0 9 11 5 10 9 10
8% 0.0 0.0 0.0 0.0 10 9 7 5
10% 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Table 25. Effect of adding 21 amino acid on crude bacteriocin.
E. coli B. subtilis
21 amino
acid
Strains
L . fermentum
10
L. acidophilus
110
L . acidophilus L . pentosus
160
L . fermentum
10
L. acidophilus
111 110
L . acidophilus
111
L . pentosus
160
2% 8 0.0 0.0 9 13 12 11 0.9
4% 7 5 7 10 0.0 0.0 0.0 0.0
6% 0.0 11 9 0.0 7 5 6 0.0
8% 11 10 7 0.0 0.0 0.0 0.0 0.0
10% 9 0.0 0.0 10 9 7 5 0.0

Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.
Isolated from Some Dairy Products under Different Culture Conditions 355
Table 26. Effect of adding some vitamins on crude bacterio-
cin.
B12 B comple
Vitamins
Strains E. coli B. subtilis E. coli B. subtilis
L . fermentum
10 0.0 11 14 11
L. acidophilus
110 0.0 10 13 5
L . acidophilus
111 10 11 14 11
L
. pentosus
160 0.0 11 10 5
4. Conclusions
Bacteriocin production was strongly dependent on pH,
nutrients source and temperature various physicochemi-
cal factors seemed to affect bacteriocin production as
well as its activity.
The bacteriocin suspension of Lactobacillus spp. grown
in MRS broth had the best inhibitory effect against wide
spectrum of bacteria. The present study demonstrated the
production of the bacteriocin by four lactobacilli isolates
under different culture conditions. Its antimicrobial po-
tency, pH stability, activity retention in low and high
temperatures suggested its wide applicability in acidic
pH conditions and in pre-processed food products. Fur-
ther research though, should be performed to develop
extraction techniques for lactic acid and bacteriocins and
test further their production on the nutrient media.
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