Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp . Isolated from Some Dairy Products under Different Culture Conditions

Lactobacilli belong to the group of lactic acid bacteria (LAB), that have several distinguished abilities such as production of lactic acid, enzymes such as β-Galactosidase and natural antimicrobial substances called bacteriocins. Bacteriocin 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 Lactobacillus spp. potential for producing 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 bacteriocins 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.


Introduction
Lactic acid bacteria (LAB) produce a number of antimicrobial substances such as organic acids, free fatty acids, ammonia, reuterin, diacetyl, hydrogen peroxide and bacteriocin, which have the capacity to inhibit the growth of food spoilage and pathogenic organisms [1].Bacteriocins are proteinaceous and ribosomally synthesized antibacterial 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 discovery, 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 foodborne 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 widespread 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 protection.Quite recently a new classification has been proposed by Cotter et al. [6].In this scheme the most dramatic 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 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-lanthionine containing and membrane-active peptides (<10 kDa).The class III bacteriocins, have been found in Lactobacillus, include heat labile proteins of large molecular mass.The class IV bacteriocins are a group of complex proteins, associated with other lipid or carbohydrate moieties, which appear to be required for activity.They are relatively hydrophobic and heat stable [8].Different bacteriocin exhibits different inhibition profile on food spoilage and pathogenic microorganisms.Therefore, they could be natural replacements for synthetic 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 fermentation conditions, such as pH, temperature and agitation [9].
The optimization of bacteriocin production and enhancement of its activity are economically important to reduce the production cost.Thus, the aims of this study were to formulate industrial media for bacteriocin production by four lactic acid bacteria isolates and the optimization of culture conditions for maximizing bacteriocin production.

Isolation and Identification of Lactic Acid Bacteria
The lactic acid bacteria were isolated from raw milk and Ras cheese, by appropriate dilutions with NaCl physiological.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 Lactobacillus spp and incubated at 37˚C for 48 -72 h [10].
The strains were sub-cultured onto MRS agar slant incubated at 30˚C for 24 h and preserved in 20% glycerol at −80˚C.One of the isolates was selected for further studies.It exhibited strong inhibitory activity against indicator 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 characteristics of the lactobacilli as described in Bergey's Manual of Determinative Bacteriology [11][12][13][14] and fermentation 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).

Treatment of Bacteria Prior to Production of Bacteriocin
The isolates were tested for their production of Bacteriocin.E. coli ATCC 25922 and Bacillus subtilis NCIB3610 were used as indicator microorganism in all assays.Indicator microorganisms used are propagated for 48 h in the Nutrient agar media, and at the temperatures indicated 30˚C.

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 subtilis NCIB3610 was evaluated by deferred methods: 1) welldiffusion assay [15] and 2) Tetrazolium/formazan-test method [16].

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 adjusting 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.

Influence of Medium Component on the Production of Bacteriocins
The effect of medium ingredients on bacteriocin production was evaluated using composed media.The supplements studied were tryptone, yeast extract, beef extract, triammonium citrate sodium acetate, MgSO 4 -7H 2 O, K 2 HPO 4 , 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 supernatant through a 0.2 ml pore-size cellulose acetate filter.

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.
Broth media were used as seed culture (10% of the total volume of the fermentation medium).The culture was adjusted to pH 7.0.

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 bacteriocins, a cell-free solution was obtained by centrifuging (6000 rpm for 20 min.at 4˚C) the culture and was adjusted to pH 7.0 [20,21].

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 activity was detected against E. coli and Bacillus subtilus at each of these temperatures [22] by teterazoluim chloride method.

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 determined against the indicator organism by agar-well diffusion assay.

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 bacteriocin preparation (positive control).All samples were incubated at room temperature for 2 hours then tested for residual antimicrobial activity by teterazolium formazan test.

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 temperature for 2 hours and tested for residual antimicrobial ac-tivity by teterazolium formazan test.

Effect of Metal Ions on Crude Bacteriocins
Activity In a separate experiment the effect of metal salts on bacteriocin was examined by adding AgNO 3 , CuSO 4 , FeSO, MgSO 4 , MnCl 2 , and ZnSO 4 (Merck) to 10 ml of crude bacteriocin preparation (0.5% final concentration).Untreated 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.

Effect of Different Concentration of NaCl on
Crude Bacteriocins Activity In a separate experiment the effect of different concentration 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 temperature for 2 hours and tested for residual antimicrobial activity [24] by agar-well diffusion assay.

Effect of Different Concentration of Amino
Acids on Crude Bacteriocins Activity In a separate experiment the effect of different concentration 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 preparation.Untreated bacteriocin preparation (positive control).All samples were incubated at room temperature for 2 hours and tested for residual antimicrobial activity by agar-well diffusion assay.

Effect of Different Concentration of Vitamins
on Crude Bacteriocins Activity In a separate experiment the effect of different concentration 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 incubated at room temperature for 2 hours and tested for residual antimicrobial activity by agar-well diffusion assay.

Statistical Analysis
Data are presented as the mean ± standard deviation, and n represents the number of the isolates and the control.

Isolation and Identification of Bacteriocinogenic Strains
Sixteen isolates of LAB were isolated from the samples.
After series of purification on MRS agar, four isolates were found to be Gram-positive, catalase negative, nonmotile bacilli.In addition, all strains were tested for growth at 10˚C for 10 days, 45˚C for 48 h, and CO 2 production 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. acidophilus CH1, L. fermentum M1 and L. pentosus CH2.

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 preservation [26].Application of bacteriocins may help reduce the use of chemical preservatives and/or the intensity of heat and other physical treatments, satisfying the demands 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. acidophilus 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. acidophilus 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 species.The present L. pentosus CH2 isolate showed inhibitory activity against Bacillus 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 confirmed that the bacteriocins of Gram-positive bacteria generally exhibit antagonistic activity against Gram-positive 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 spectrum against Gram-positive and Gram-negative bacteria using the AWD method.

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 individual bacteriocins [5].Several media have been evaluated by numerous authors to improve bacteriocin synthesis [30] because these peptides are not always produced in standard or enriched culture media.Lactic acid bacteria 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 richmedium supernatant is an additional problem, making the purification of the bacteriocin a relatively complicated protocol.The present study was primarily aimed to determine cultural conditions for obtaining better and stable bacteriocins production.L. acidophilus CH1 was able to produce bacteriocins, which had a wide inhibitory spectrum towards both Gram-negative and Gram-positive food spoilage and pathogenic bacteria.Results show that bacteriocin 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.Bacteriocin 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 production of bacteriocin by Lactobacillus isolates.Table 5 showed that MRS seemed to be more suitable medium    for the bacteriocin production.Similar results were observed by [34,35].Results in Table 5 also indicate that larger amounts of the bacteriocins were synthesized only in MRS medium supplemented with K 2 HPO 4 (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 cultivation media might have an influence on the amount of bacteriocin produced by microorganisms.Similar observations have been made previously.Daba et al. [21] obtained 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.Modification of nutrients of cultivation media should be considered for maximal production of bacteriocin that has potential use as a food biopreservative [33].Similar results 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 bacteriocin from the cell surface of the producer strain.This finding was supported by the increased bacteriocin production in the medium supplemented with different concentrations of yeast extract plus Tween-80.An earlier study by the senior author [28] revealed that in L. plantaram MTCC1746, maximum bacteriocin production could be achieved by providing 1.5% yeast extract and 1.5% Tween-80.The addition of MgSO 4 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.

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 inhibition, it could also be related to the control that the supplied sugar substrate exerts on the bacteriocm biosynthesis.Biswas et al. [33] reported that MRS medium is a better medium for cell growth and bacteriocins production than other media.Generally, maximum production corresponds to against pathogenic microbe such as Bacillus subtilis NCIB3610 and E. coli ATCC 25922.Therefore increased cell concentrations in a high celldensity reactor is expected to increase bactenocm production.In general bactencin production by lactic acid bacteria occurs during the active growth phase [29,38].
Conditions favouring bacterial growth and high cell densities are frequently beneficial to bactenocin production as well [38].However, a high cell yield does not necessarily 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 between environmental conditions and bactenocin activity levels and no generalisation about the optimum conditions 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 studied to obtain a better understanding of the production mechanism.

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. 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 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 bacteriocin but the smallest of the antimicrobial activity was 39.34% in temp.90˚C/30min wherever Lactobacillus fermentum was showed antimicrobial activity against Bacillus 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 acidophilus 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.Bacteriocin wherever Lactobacillus 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. pentosus CH2 which showed the largest growth inhibition% was 85.98% in temp.60˚C/30min against Bacillus subtilis 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 production levels were identical but biomass increased with the temperature.Moreover, the lower the temperature, the higher the volumic production, particularly for mesenterocin 52 A. This indicated that bacteriocin production was stimulated by temperatures unfavorable for growth, particularly the low temperatures.

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  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 wherever 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 subtilis 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 Lactobacillus 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 wherever Lactobacillus acidophilus CH1 was showed antimicrobial activity against Bacillus subtilis 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 subtilis 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.Similar observation was made earlier in L. acidophilus [42].

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 Table 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. acidophilus 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 wherever, 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 acidophilus 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 wherever Lactobacillus acidophilus CH1 was showed antimicrobial activity against Bacillus subtilis 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 Bacillus 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.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 FeSo 4 but that showed the smallest 66.89% in MgSo 4 .Bacteriocin wherever Lactobacillus acidophilus CH1 was showed antimicrobial activity against Bacillus subtilis NCIB3610 was 75.03% in ZnSo 4 but the smallest of the antimicrobial activity was 5.41% in CuSo 4 .Table 23, in addition, the strains L. pentosus CH2 which showed the largest growth inhibi-tion% was 97.17% in FeSo 4 against Bacillus subtilis NCIB3610 but the smallest was 15.5% in ZnSo 4 wherever, the same strain was the largest growth inhibitor% 93.11% in MgSo 4 against to E. coli but the smallest of the antimicrobial activity was 13.34% in AgSo 4 .

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 Lactobacillus pentosus CH2 against to E.coli ATCC 25922 was 14 mm, L. fermentum M1 was shown15 mm against to Bacillus subtilis NCIB3610 (Table 24).Among them, bacteriocin from Lactobacillus pentosus CH2 against 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].

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 Lactobacillus 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. coli 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 different bacteriocins were shown in 2% against E. coli ATCC 25922 and Bacillus subtilis NCIB3610.

Effect of Different Vitamins Component on
Crude Bacteriocin Activity The effects of different vitamins such as B 12 and B complex on crude of bacteriocins were studied Table 26.In MRS broth, B 12 increased the activity of bacteriocins isolated from Lactobacillus acidophillus CH1 against to E. coli ATCC 25922 was 10 mm but the three other isolates 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. acidophilus M2 and L. pentosus CH2.This result was agree with Adenike et al. [45].

Conclusions
Bacteriocin production was strongly dependent on pH, nutrients source and temperature various physicochemical 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 potency, pH stability, activity retention in low and high temperatures suggested its wide applicability in acidic pH conditions and in pre-processed food products.Further research though, should be performed to develop extraction techniques for lactic acid and bacteriocins and test further their production on the nutrient media.
Data are presented as mean ± SD.
and Optimization Using New Isolates of Lactobacillus spp.Isolated from Some Dairy Products under Different Culture Conditions 355

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
Data are presented as mean ± SD.

Table 5 . Effect of adding some nutrient components in MRS media on bacteriocins production by Lactobacillus spp. isolates.
tose medium, Medium (C) Corn steep liquor medium, Medium (D) Glycerol-molasses-liquid medium for bacteriocins (Table6).A maximum growth rate were shown in MRS and CSL for all isolates and a maximum bacteriocrn activity (inhibition zone mm of Bacillus subtilis

Table 9 . Effect of temperature on crude bacteriocins.
L. acidophilus CH1

Table 10 . Effect of temperature on crude bacteriocin. L. pentosus CH2 Bacillus subtilis NCIB3610 E. coli ATCC 25922
Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.Isolated from Some Dairy Products under Different Culture Conditions 350 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, Copyright © 2013 SciRes.FNS

Crude Bacteriocins Tables 20-23 clearly
highlights of effect of different minerals such as AgNo 3 , CuSo 4 , FeSo 4 , MgSo 4 , MnCl 2 and ZnSo 4 on crud bacteriocin from four LAB isolates according to the teterazolium chloride methods.Tables Copyright © 2013 SciRes.FNS Study Bacteriocin Production and Optimization Using New Isolates of Lactobacillus spp.Isolated from Some Dairy Products under Different Culture Conditions 351