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1554
Journal of Applied Sciences Research, 9(3): 1554-1563, 2013
ISSN 1819-544X
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLES
Studies on the Production of Antimicrobial Substances Produced from Bifidobacterium
bifidum
1
Mohamed, M.I. Helal, 1Nayera, A.M. Abdelwahed, 1Aza. M. Abdel-Fattah and 2Madeha O.I.
Ghobashy
1
2
Chemistry of Natural and Microbial Products Dept., National Research Center, Dokki, Cairo, Egypt.
Biology Department, Faculty of Sciences, Tabuk University,Tabuk, Saudi Arabia
ABSTRACT
The present study was aimed to produce bacteriocin from Bifidobacterium bifidum by using low cost
materials as corn steep liquor, soybean and sugar cane molasses by the study of the influence of different
environmental factors as temperature, pH and incubation time. Data revealed that the optimized conditions for
the production of bacteriocin was by growing Bifidobacterium bifidum on MRS broth medium with lactose 2 3% instead of glucose with initial pH 6.5 , incubation temperature 37 °C and incubation time of 72 hours to
reach inhibition zone of 28, 29 and 30 mm. On the other hand sucrose and cane sugar molasses gave negative
results for cell growth and bacteriocin production. The effect of low cost proteins like soybean gave very weak
results of bacteriocin production but gave high growth of B.bifidum cells. On other hand corn steep liquor gave a
moderate production for both cells and bacteriocin near the control results.
Key words:
Introduction
Probiotics are defined as selected, viable microbial dietary supplements that when introduced in sufficient
quantities, beneficially affect human organism through their effects in the intestinal tract (Dimer & Gibson,
1998; Zimmer & Gibson, 1998; Sanders, 1998; Vaughan et al., 1999; Zubillaga et al., 2001and Holzapfel &
Schillinger, 2002). There are a large number of probiotics currently used and available in dairy fermented foods,
especially in yogurts. Lactic acid bacteria constitute a diverse group of organisms providing considerable
benefits to humankind, some as natural inhabitants of the intestinal tract and others as fermentative lactic acid
bacteria used in food industry, imparting flavor and texture and possessing preservative properties. Beyond
these, some species are administered to humans as live microbial supplements, which positively influence our
health mainly by improving the composition of intestinal microbiota. For this reason, they are called probiotics.
Some selected strains of Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus and Saccharomyces have
been promoted in food products because of their reputed health benefits (Dimer & Gibson 1998; Sanders, 1998;
Fuller, 1991; Ouwehand et al., 1999 and Puupponen-Pimia et al., 2002).Lactic acid bacteria (LAB) are a group
of bacteria that can preserve dairy foods by producing a number of organic compounds that are antagonistic to
other microorganisms (Lindgren and Dobrogosz, 1990). Among these compounds proteinaceous-bacteriocins
have gained much attention especially regarding their role in the diary foods where they are known to strongly
inhibit the growth of pathogens (Ruiz-Barbara et al., 1994; Benkerroum et al., 2007). Much research has
focused on utilizing bacteriocins as novel food preservatives, but there is also interest in using them for the
control of bacterial diseases in humans and animals. The probiotic potential of these bacteria is also vastly
investigated by Gilliand, 1990; Cleveland et al., 2001; Mojgani and Ashtiani, 2006 and Diez-Gonzalez, 2007.
Production of bacteriocins can be influenced by many things, including medium composition (Zhou et al.,
2008), environmental factors (Leal-Sánchez et al., 2002; Motta and Brandelli, 2008), and other growth
conditions. De Man Rogosa and Sharpe (MRS) is the standard culture media for lactic acid bacteria (LAB), but
its high cost limits its suitability for industrial-scale production. MRS has ever been used for large scale
fermentation, while the purpose was to produce something, like some enzymes, or other metabolin (Hummel et
al., 1983 and Lu et al., 2003). Studies lowering the cost of culture media have been published (Dominguez et
al., 2007; Trinetta et al., 2008 and Wiese et al., 2010), not referring to industrial fermentation. The objective of
the present study was to increase the efficiency of bacteriocin production and reduce the cost of fermentation,
thereby increasing the viability of industrial scale production.
Corresponding Auther: Dr. Mohamed M.I. Helal, Chemistry of Natural and Microbial Products Dept., National Research
Center, Dokki, Cairo, Egypt.
E-mail: [email protected]
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J. Appl. Sci. Res., 9(3): 1554-1563, 2013
Materials and Methods
Bacterial Strains:
Bifidobacterium bifidum (B.bifidum) which is Gram-positive bacillus shaped bacterium kindly provided
from Chr. Hansen's Lab. Inc., Denmark. Pathogenic strains, Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa and Staphylococcus aureus were obtained from the clinical lab. of the Nozha
International Hospital in Cairo, Egypt.
Positive control of antibiotics:
It´s include Streptomycin (15 µg) as antibacterial antibiotic and Fluconazole (150 µg) as antifungal
antibiotic derived from pfizer pharmaceutical company.
Sugar cane molasses by product obtained from "The Sugar and Integrated Industries Co.", El Hawamdia,
Giza, Egypt.
Corn steep liquor (CSL) by product was obtained from Egyptian Starch & Glucose Company,
Moustoroured, Egypt.
Soybean meal -extract (SBE) prepared by aqueous extraction (at 121ºC for 20 min.) of a commercial
sample of soybean seeds (solid/liquid ratio 1/20, w/v) followed by filtration and freeze drying of the resulted
extract.
Gas Generating Kits used for generation of hydrogen and carbon dioxide in metal or plastic anaerobic jars.
It was purchased from Oxoid Ltd, Basingstoke, Hampshire, England.
Culture Media:
a- Nutrient agar medium was used for the growth and maintenance of the pathogenic strains Escherichia
coli , Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus.
b- MRS medium (De Man- Rogosa- Sharp- medium was used for the growth and maintenances of the
probiotic B. bifidum. it was prepared without addition of agar and can be used for determination of the growth
density of the investigated probiotic and its bacteriocin production.
c- Potato Dextrose Agar (PDA): Was used for the growth and maintenances of the yeast strain Candida
albicans.
Isolation and purification of bacteriocin:
Variations in the level of bacteriocin production were evaluated by the well diffusion assay (Schillinger and
Lücke, 1989) and using cork pore diameter 5 mm. Experiments were carried out in duplicate. 0.1 % (v/v) of B.
bifidum was inoculated at a final concentration of about 107 CFU/ml. The antimicrobial activity of the
supernatants was evaluated by the critical dilution assay of Barefoot and Klaenhammer (1983) with using of
Streptomycin (15 µg) as antibacterial antibiotic and Fluconazole (150 µg) as antifungal antibiotic. Bacteriocin
activity was defined as the reciprocal of the highest dilution showing definite inhibition of the indicator strains
and was expressed as activity units per milliliter (AU/ml).
Determination of the minimum inhibitory concentration (MIC) of B.bifidum supernatent on indicator pathogens:
According to Andrews et al., 2001, minimum inhibitory concentration (MIC) is the lowest concentration of
an antimicrobial metabolite that inhibit the visible growth of a microorganism after overnight incubation.
Supernatants obtained from B. bifidum bacterium was used to determine the MIC of each by using the original
supernatant of each as follow: 0.025, 0.05, 0.10, 0.15, 0.20 and 0.25 ml . Well-cut diffusion technique was used
to determine the minimal inhibitory effect of the previous dilutions against the indicator pathogens.
Preparation of cell-free culture supernatants:
Cell-free culture supernatants (CFCS) were obtained by centrifugation at 5000 rpm for 20 min of B. bifidum
culture grown under specific cultivation conditions under study.
Optimization studies:
Incubation temperature ranged from 25-45ºC and initial pH ranged from 5.5 to 8.0. Effect of various carbon
sources and nitrogen sources were evaluated in relation to bacteriocin production.
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3- Results:
3.1. Culture conditions for bacteriocin production:
3.1.1. Effect of incubation temperatures:
Results revealed that the optimum temperature for the production of bacteriocin was from 35 to 37°C at
incubation time of 72 hours and the highest activity against pathogenic microorganisms expressed by the
inhibition zone diameter was 24 mm for Escherichia coli, 25 mm for Pseudomonas aeruginosa, 23 mm for
Klebsiella pneumoniae, 27 mm for Staphylococcus aureus and 23 mm for Candida albicans table (1).
Streptomycin antibiotics control gave 20 mm inhibition zone diameter at 37°C for all pathogenic bacterial
strains after 24 hrs., while Fluconazole control gave 14 mm inhibition zone diameter for Candida albicans at
28°C for 48-72 hrs. Growth of Bifidobacterium bifidum caused high levels of turbidity in MRS broth medium
(OD 600>1). Optimum temperatures from 32 – 37oC and incubation time 72 hours for the highest cell growth
expressed by the optical density was represented in figure (1).
Table 1: The effect of different incubation temperatures values on bacteriocin activity as inhibition zone (mm).
E.coli
Pseudomonas
Klebseilla pneumonia
Staph. Aureus
aeuroginosa
24h
48h
72h
24h
48h
72h
24h
48h
72h
24h
48h
72h
25°C
10
12
13
14
14
15
12
12
13
-ve
-ve
13
30°C
15
16
23
17
23
24
14
17
20
14
18
25
32°C
16
16
23
17
24
24
15
17
20
14
15
25
35°C
16
16
24
17
24
25
15
17
23
16
19
27
37°C
16
16
24
17
24
25
15
17
23
16
19
27
40°C
12
13
13
11
15
17
11
12
18
11
12
16
45°C
12
12
12
10
10
11
11
12
11
10
11
12
3.0
Candida albicans
24h
11
11
14
14
15
10
10
48h
12
14
15
15
15
11
11
72h
12
23
19
23
23
11
10
24 hours
48 hours
72 hours
2.5
Optical density
2.0
1.5
1.0
0.5
0.0
25
30
32
35
37
40
45
0
Temperature C
Fig. 1: The effect of different incubation temperatures on Bifidobacterium bifidum cell growth.
3.1.2 Effect of different pH values:
Table (2) and figure (2) revealed that the optimum pH was 6.5 for the production of bacteriocin as well as
cell growth at incubation time of 72 hours and the highest activity against pathogenic microorganisms expressed
by the inhibition zone diameter 24 mm for Escherichia coli, 25 mm for Pseudomonas aeruginosa, 23 mm for
Klebsiella pneumoniae, 27 mm for Staphylococcus aureus and 19 mm for Candida albicans also maximum OD
was 2.873 at the same pH(6.5).
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J. Appl. Sci. Res., 9(3): 1554-1563, 2013
Table 2: The effect of different pH values on bacteriocin activity as inhibition zone (mm).
E.coli
Pseudomonas
Klebseilla pneumonia
Staph. Aureus
aeuroginosa
24h
48h
72h
24h
48h
72h
24h
48h
72h
24h
48h
5.5
10
12
13
14
14
15
12
12
13
-ve
-ve
6.0
13
14
15
16
17
17
13
13
15
11
13
6.5
16
16
24
17
24
25
15
17
23
16
19
7.0
16
16
22
17
24
25
15
17
20
16
19
7.5
15
15
16
17
20
20
13
13
16
17
17
8.0
12
13
13
11
15
17
11
12
12
11
12
3.0
Candida albicans
72h
13
19
27
27
18
12
24h
11
12
15
14
13
10
48h
11
13
15
15
13
11
72h
12
15
19
19
14
11
24 hours
48 hours
72 hours
2.5
Optical density
2.0
1.5
1.0
0.5
0.0
5.5
6.0
6.5
7.0
7.5
8.0
Initial pH
Fig. 2: The effect of different pH on Bifidobacterium bifidum cell growth.
3.1.3 Effect of different carbon sources:
The effect of different glucose concentrations on the bacteriocin production can be shown in table (3). An
increase in bacteriocin activity occurred until reached to 2% glucose concentration after 72 hr. incubation.
Above this concentration, a slight decrease of the bioactive metabolite was noticed. On the other hand figure (3)
show that the maximum growth occurred at 3% glucose (O.D 2.93). The effect of replacement of glucose by
lactose (1-4%) at different incubation time (24-72hr.) illustrated at table (3). By using lactose concentration 3%
and incubation time of 72 hours, highest bacteriocin activity against Candida albicans was recorded 30 mm
inhibition zone, followed by Escherichia coli which its inhibition zone diameter 29 mm and 24 mm against
Pseudomonas aeruginosa was attained whereas maximum bacteriocin activity against Klebsiella pneumoniae is
fixed for all lactose concentrations from 1-4% after 48hr. Bacteriocin activity produced in media containing
lactose 4% for 72hr gave inhibition zone diameter of 28 mm against Staphylococcus aureus. Concerning cell
growth of the producer strain Bifidobacterium bifidum optimum condition for growth was attained by using 3%
lactose concentration and incubation time of 72h. Above this concentration the cell growth remains constant as
shown in figure (4).
By using of different sucrose concentrations as carbon source instead of glucose, low bacteriocin activity
obtained compared with glucose and lactose (table 3), with relatively low cell biomass (O.D of 2.046 at 72hrs
and 1% sucrose concentrations figure 5). The effect of replacement of glucose by sugar cane molasses was
tested (table 3). Low bacteriocin produced against pathogenic microorganisms except for Escherichia coli when
sugar cane molasses was used at 2% concentrations and 72hours gave fixed bacteriocin activity (23 mm) .High
cell biomass (O.D. reach 3.818) at 48hr occurred on using 4 % sugar cane molasses figure (6).
3.1.4. Effect of different cheap protein sources:
3.1.4.1 Effect of soybean meal concentrations:
Table (4) discuss the using of different soybean meal concentrations as a nitrogen source for bacteriocin
production (instead of all nitrogen sources in the fermentation medium,peptone 10%, meat extract 8% and yeast
extract 4%). The bacteriocin activity against all pathogens was low ranged from 10-12 mm inhibition zone
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diameter and negative values were recorded against Candida albicans, however cell biomass at 72 hours and 4%
soybean meal concentrations reached OD 0.981 diluted 100X (figure 7).
Table 3: The effect of different carbon sources concentrations on bacteriocin production by B. bifidum as inhibition zone (mm).
E.coli
Pseudomonas
Klebseilla
Staph. Aureus
Candida albicans
aeuroginosa
pneumonia
Carbon
source
24h
48h
72h
24h
48h
72h
24h
48h
72h
24h
48h
72h
24h
48h
72h
glucose
1%
16
16
16
18
20
21
12
12
18
13
14
20
13
14
16
2%
16
16
24
17
24
25
15
17
23
16
19
27
15
15
23
3%
16
16
19
18
25
25
12
17
20
16
19
27
14
15
20
4%
15
16
18
16
22
22
12
17
19
17
18
25
15
15
19
lactose
1%
17
18
14
16
22
19
13
20
17
14
20
15
15
23
25
2%
16
20
27
17
22
21
17
20
17
21
20
20
17
22
28
3%
16
24
29
21
22
24
13
20
17
25
20
20
17
22
30
4%
16
23
26
19
22
23
13
20
17
16
21
28
17
22
30
sucrose
1%
15
17
18
18
20
20
20
20
20
17
19
20
10
11
11
2%
18
20
20
18
20
20
20
20
20
18
18
20
10
12
12
3%
17
20
20
20
20
20
20
21
21
18
19
20
12
11
11
4%
18
20
20
21
20
21
20
21
21
19
19
20
11
11
11
sugar cane molasses
1%
15
21
21
18
18
18
14
16
17
15
16
17
10
10
10
2%
15
23
23
18
16
17
14
15
17
15
16
16
10
12
12
3%
15
17
17
17
17
18
14
16
17
15
16
17
12
12
12
4%
16
17
18
17
18
18
15
16
17
14
16
17
11
12
12
Table 4: The effect of different protein sources concentrations on bacteriocin production by Bifidobacterium bifidum as inhibition zone
(mm).
Protein
E.coli
Pseudomonas
Klebseilla pneumonia
Staph. Aureus
Candida albicans
source
aeuroginosa
24h
48h
72h
24h
48h
72h
24h
48h
72h
24h
48h
72h
24h
48h
72h
Soy bean %
1
10
10
11
10
10
10
10
11
12
10
11
12
-ve
-ve
-ve
2
11
12
12
11
11
12
11
12
12
11
11
12
-ve
-ve
-ve
3
10
11
12
10
11
12
10
12
12
10
11
12
-ve
-ve
-ve
4
10
11
12
10
11
12
10
12
12
10
12
12
-ve
-ve
-ve
Corn steep liqour %
1
20
18
17
30
18
17
20
18
17
20
19
19
16
17
17
2
21
24
22
31
24
22
21
24
22
22
23
23
18
20
20
3
20
24
22
30
24
22
21
24
22
21
23
22
18
20
20
4
20
24
22
30
24
22
21
24
22
22
23
22
18
20
20
3.0
24 hours
48 hours
72 hours
Optical density
2.5
2.0
1.5
1.0
0.5
0.0
1%
2%
3%
4%
glucose concentration %
Fig. 3: The effect of different glucose concentrations on Bifidobacterium bifidum cell growth.
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3.0
24 hours
48 hours
72 hours
Optical density
2.5
2.0
1.5
1.0
0.5
0.0
1%
2%
3%
4%
lactose concentration %
Fig. 4: The effect of different lactose concentrations on Bifidobacterium bifidum cell growth.
2.0
Optical density
24 hours
48 hours
72 hours
1.5
1.0
0.5
0.0
1%
2%
3%
4%
sucrose concentration %
Fig. 5: The effect of different sucrose concentrations on Bifidobacterium bifidum cell growth.
3.1.4.2 Effect of different Corn Steep Liquor concentrations:
By using corn steep liquor as a nitrogen source instead of all nitrogen sources in the fermentation medium
(peptone 10%, meat extract 8% and yeast extract 4%) influence on the activity of the bacteriocin in all
concentrations as shown in table (4). Production of bacteriocin was optimum at 2% corn steep liquor
concentration for all the pathogenic microorganisms.
High cell biomass (O.D. 0.981 diluted 10X) was recorded at 72hrs and 4% corn steep liquor concentrations
figure (8).
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4.0
24 hours
48 hours
72 hours
3.5
3.0
Optical density
2.5
2.0
1.5
1.0
0.5
0.0
1%
2%
4%
3%
sugar cane molasses concentration %
Fig. 6: The effect of different cane sugar molasses concentrations on Bifidobacterium bifidum cell growth.
1.0
0.9
24 hours
48 hours
72 hours
Optical density
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1%
2%
3%
4%
soybean meal concentration %
Fig. 7: The effect of different soy bean meal concentrations on Bifidobacterium bifidum cell growth (diluted
100x).
3.1.4.2 Minimum Inhibitory Concentration (MIC) Test:
Table (5) indicated that 0.05 ml of the culture filterate containing bacteriocin is the volume above which
negative effect of bacteriocin against bacteria and Candida was recorded. It was considered as the minimum
inhibitory concentration MIC. On other hand above this volume of culture filtrate highest activity of bacteriocin
against pathogenic microorganisms was recorded i.e. 0.15 ml as well as 0.2 ml and 0.25 ml of culture filtrate is
the best concentrations gives the highest bacteriocin yield and activity against all strains.
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Table 5: The effect of MIC values on bacteriocin production as inhibition zone (mm).
Culture
Escherichia coli
Pseudomonas
Klebsiella
filtrate
aeuroginosa
pneumoniae
conc.(ml)
24h
48h
72h
24h
48h
72h
24h
48h
72h
0.025
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
-ve
0.05
10
11
11
13
13
13
11
13
13
0.10
13
15
15
16
18
18
14
14
14
0.15
16
16
18
17
24
25
15
17
20
0.20
16
16
17
17
25
25
15
17
21
0.25
16
16
17
18
25
25
16
17
21
Staphylococcus
aureus
24h
48h
72h
-ve
-ve
-ve
10
12
12
14
16
16
14
15
27
14
16
20
15
17
20
Candida albicans
24h
-ve
12
15
14
15
15
48h
-ve
14
16
15
15
15
72h
-ve
14
16
19
19
19
1.0
0.9
24 hours
48 hours
72 hours
Optical density
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1%
2%
3%
4%
corn steep liquor concentration %
Fig. 8: The effect of different corn steep liquor concentrations on Bifidobacterium bifidum cell growth (diluted
10x).
Discussion:
Effects of physical factors on bacteriocin production, including temperature, pH and incubation time were
recently studied (Delgado et al., 2007). The composition of the medium was also shown to have an important
role in bacteriocin production (Li et al., 2002). However, studies to reduce the cost of the medium have only
been recently conducted (Dominguez et al., 2007). In the present study, some nitrogen sources in MRS were
replaced to reduce costs were used.
Environmental factors are the key parameters that have pronounced influence on the growth rate and
magnitude of formation of antibacterial substances by bacteria. Furthermore, in case of bacteriocin, production
is strongly dependent on medium composition (Gänzle et al., 1999). Also pH 6.5 is the optimum pH value gives
the highest bacteriocin productivity and considerable cell growth of the studied probiotic., Leroy and De Vuyst,
2002 reported that the bacteriocin activity of B. bifidum was lower at pH ranging from 7.5 to 8.0 than pH in the
range 5.5 – 6.5. Our results agree with Todorov and Dicks, 2005 who reported that low level of bacteriocin
obtained at pH 6 or less.
Generally, bacteriocin production by Bifidobacterium bifidum was reported as a temperature sensitive
process. On the other hand Leroy and Vuyst ,1999b reported that the optimal temperature for bacteriocin
production does not necessarily coincide with the optimal growth temperature. It has been suggested that
bacteriocin production by Bifidobacterium bifidum is enhanced by suboptimal temperatures (De Vuyst et al.,
1996). However, in the present study, results showed that temperatures ranged from (30 – 37 °C) was optimum
for Bifidobacterium bifidum growth determined a lower production of antimicrobial compounds. A higher
production of bacteriocin in the range of temperature for optimal strain development has been observed in many
LAB strains, such as E. faecium RZS C5 (Leroy and De Vuyst, 2002), Lactobacillus sakei Lb 706 (Schillinger
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J. Appl. Sci. Res., 9(3): 1554-1563, 2013
and Lücke, 1989) and Lb. sakei CTC 494 (Leroy and De Vuyst, 1999a), and it has also been reported for
jenseniin G produced by Propionibacterium thoeni (jensenii) P126 (Ekinci and Barefoot, 2006). No significant
differences were found for bacteriocin production yield at 30 and 37 °C, suggesting that growth temperature in
that range does not play a defining role in bacteriocin activity. The same observation was reported by Todorov
and Dicks (2005) with regards to Lactobacillus plantarum bacteriocins.
By testing the effect of different nutritional factors on bacteriocin production derives from the need of
selecting compounds that are basic for bacteriocin biosynthesis and for attaining higher yields, these substrates
may be added to minimize cost media, such as wastes from the food industry (e.g. molasses, corn steep liquor)
or low cost protein soy bean, in order to reduce large-scale production costs.
With regards to studied carbohydrates, low bacteriocin production was produced when using sucrose and
sugar cane molasses. Since both molasses and sucrose reduced cell biomass, we inferred that the bacteria could
not make good use of these saccharides, although monosaccharide were readily utilized (glucose and lactose).
Bing, et al., 2011observed that the concentration of glucose over 2.0 g/l could possibly reduce the bacteriocin
yield. On other hand, nitrogen source soy bean reduce or inhibit completely the bacteriocin production but the
cell growth is too high. It explain that the studied probiotic B. bifidum use the soy bean to produce cells only not
for bacteriocin production while corn steep liquor gave low bacteriocin production with moderately high cell
growth although several authors reported that higher bacteriocin activity were observed with increased nitrogen
concentrations (Parente and Hill, 1992; Vignolo et al., 1995; Aasen et al., 2000; Guerra and Pastrana, 2001).
Conclusion:
From the previous results we can conclude that bacteriocin activity and cell growth of B. bifidum was
strongly affected by the initial pH, incubation temperature, nitrogen and carbon sources. The best optimized
condition was by growing on MRS broth but with lactose 2% or 3 %, with initial pH 6.5 and 37 °C incubation
temperature, and the minimum inhibitory concentration of culture filtrate free from cells was 0.15 ml to inhibit
the growth of the pathogenic indicators.
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