Lactic acid bacteria (LAB) are Gram positive, non-spore forming, catalase negative cocci or fermentative lactobacilli which produce lactic acid from fermentation of carbohydrates [1,2]. These bacteria are the major component of the starters used in fermentation, especially for dairy products, and some of them are also natural components of the gastrointestinal microflora. Lactobacillus is one of the most important genera of LAB [3,4]. These organisms are also known to produce various compounds such as bacteriocin which can antagonize the growth of some pathogenic bacteria in foods [5,6]. Lactic acid bacteria are regarded as a major group of probiotic bacteria and have been used successfully to treat acute infantile diarrhea and various diarrheal illnesses [7,8].

They are considered as generally recognized as safe (GRAS) organisms and can be safely used as probiotics for medical and veterinary applications [3,9]. Probiotics are beneficial bacteria as they promote good digestion, boost immune function, increase resistance to infection, inhibit the growth of harmful bacteria and favor the intestinal microflora balance alteration [3,10]. There are also other physiological benefits of probiotics has been published as it help in removal of carcinogens, lowering of cholesterol, immunostimulating and allergy lowering effect, synthesis and enhancing the bioavailability of nutrients, alleviation of lactose intolerance [11].

Another study showed the effects of Lactobacillus species on the disease severity, of children with atopic dermatitis and concluded that supplementation of a probiotic is associated with clinical improvement in children with atopic dermatitis [12].

Variety of microorganisms including yeasts, molds and bacteria are present in raw milk. However, among these organisms, only the lactic acid bacteria (LAB) have the property of producing lactic acid from milk sugars by the process of fermentation and thus LAB constitute the predominant microflora of milk [13]. These lactic acid bacteria called probiotic and, it is defined classically as a viable microbial dietary supplement that beneficially affects the host through its effects in the intestinal tract. Probiotics are viable lactic acid microorganisms that are believed to provide health benefits when administered in appropriate quantities [14]. The lactic acid bacteria are known to produce antibacterial substances including bacteriocins which can inhibit the growth of several pathogenic bacteria.

Bacteriocins are small ribosomally synthesized peptides that are active against other bacteria and against which the producer has a specific immunity mechanism [15]. Bacteriocins are a heterogeneous group and are usually classified into peptides that undergo significant post-translational modifications (class I) and unmodified peptides (class II) [16]. Bacteriocins from lactic acid bacteria are considered safe additives, useful to control the frequent development of pathogens as broad range of antibacterial activity [17].

These bacteriocins produced by Lactic acid bacteria (LAB) are potent bio-preservative agents and the applications of these in food are currently the subject of extensive research. Finding for new bacteriocins with a wider spectrum of activity and compatibility with different food system is being studied. The present study aimed to investigate the antimicrobial properties of bacteriocins produced by Lactobacillus species isolated from raw milk.

Isolation of Lactobacillus species

Raw milk of cow, buffaloes, goat and sheep were collected from the local dairy farms of Giza, Egypt during 2011-2013. The samples were collected in a sterile screw cap tube, freshly isolated cultivated on de Man Rogosa Sharpe (MRS) medium and incubated anaerobically at 37°C for 48 hr.

The samples were collected in a sterile screw cap tube and these freshly isolated samples were cultivated on de Man Rogosa Sharpe (MRS) medium (Oxoid, Canada) by incubating anaerobically at 37°C for 48 hr. The suspected colonies were identified on the basis of Gram’s staining, catalase test, oxidase, indole, nitrate reduction, and ability to utilize different sugars according to Williams [18]. Finally, the isolates were sub cultured onto MRS agar slants which were incubated at 37°C for 24 h and preserved in 20% glycerol (Oxoid, Canada) at -20°C until further used.

Indicator isolates

The indicator bacteria, Staphylococcus aureus , Escherichia coli , S. xylosus , S. uberis and Yersinia enterocolitica were kindly supplied from Microbiology Department, Faculty of Veterinary Medicine Cairo University.

Production of crude bacteriocin

Each strain of Lactobacillus isolate was propagated in MRS broth (1000 ml) seeded with 10% inoculum (108 cfu/ml) of overnight culture and incubated for 48 h at 150 rpm at 37°C. After incubation, the whole broth was centrifuged at 10,000 × g for 15 min pH values of supernatants were adjusted to pH 6.5-7.0 by the addition of 1 N NaOH. The supernatants were membrane filtered (Millipore, 0.22 μm) and stored at 4°C [19]. The cell-free supernatant (crude bacteriocin) was characterized by SDS–PAGE as described by Pot et al. [20].

Purification of bacteriocin

The cell-free culture supernatant (crude bacteriocin) was saturated with 70% ammonium sulfate (Carl Roth, Germany) and kept at 4°C for 4-5 hrs to precipitate out the proteins. After precipitation, the pellet was collected by centrifugation at 10,000 × g for 30 min at 4°C. The pellet was were re-suspended in 25 ml of 0.05 M potassium phosphate buffer (pH 7.0) and dialyzed in a tubular cellulose membrane (Specrapor, 1000 dalton MWco, Fisher Scientific Pittsburgh, PA, USA) against 2 litres of the same buffer at 4°C overnight in spectrapor No. 4 dialysis tubing [19]. The dialyzed protein was applied to a Sephadex G-100 column (1.6 cm × 36 cm) (GE Healthcare, Germany) pre equilibrated with phosphate buffer (pH 7.0). The flow rate was adjusted to 24 ml/h and fractions (1 ml each) were collected in tubes. The collected fractions showing high bacteriocin activity were pooled and lyophilized using Lypholizer (Thermo Fisher Scientific, Germany).

Bacteriocin assay

The antibacterial activity of the bacteriocin extracted from Lactobacillus strains was determined using the agar well diffusion method as described by Ivanova et al. [21]. 50 μl of the bacteriocin were placed in 5 mm diameter wells that had been cut in agar plates previously seeded with 106 cfu/ ml of the indicator bacteria. After incubating the plates for 24 hrs at 37°C, the diameter of zone of growth inhibition was measured. Antimicrobial tests were done in duplicate and the mean values were recorded.

Characterization of bacteriocins

Bacteriocins were characterized mainly on the basis of effect of pH and effect of temperature as suggested by Ivanova et al. [21]. The purified bacteriocin from different lactobailli isolates had variable molecular weight (M.Wt).

Effect of pH

To determine effect of pH, 0.5 ml of purified bacteriocin was added into 4.5 ml of nutrient broth at different pH values (3 to 10) and incubated for 30 min at 37°C. Each of the bacteriocin samples treated at different pH values was assayed against indicator bacteria by well diffusion method.

Effect of temperature

Purified bacteriocin (0.5 ml) was added into 4.5 ml of nutrient broth in the test tube. Each test tube was then overlaid with paraffin oil to prevent evaporation and then heated at different temperatures (40, 60, 80 and 100) °C for 10 min. The bacteriocin activity of different heattreated was measured by well diffusion method.

Identification of Lactobacillus species among the examined milk samples

Different Lactobacilli were isolated from the collected milk samples as shown in Figure 1 and listed in Table 1A and 1B, L. acidophilus , L. salivarius and L. delbrueckii subsp. bulgaricus isolates were specifically detected from cow milk samples. Whereas, L. acidophilus , L. fermentum and L. pentosus isolates were detected from buffalo milk samples. Moreover, L. acidophilus , L. rhamnosus and L. delbrueckii subsp. bulgaricus isolates were detected from ewe milk sample. However, L. helveticus and L. brevis isolate were detected from goat milk.

Figure 1: (I) microscopically appearance of lactobacilli stained with Gram's stain (100x). A. L. delberuckii subsp. bulgaricus B. L. brevis C. L. pentosus D. L. salivarius F. L. rhamnosus. (II) characteristic colonies of lactobacilli on MRS medium. A. L. brevis large white colony B. L. acidophilus medium size white colony.

Table 1A: Identification of Lactobacillus species among the examined milk samples.

Table 1B: Bacteriocin activity of Lactobacillus isolates among the indicator bacteria.

Bacteriocins of L. delbrueckii subspecies bulgaricus having M.Wt (16.5 and 21 kDa) isolated from cow and ewe showed inhibition zone against the indicator bacteria. Bacteriocin of L. brevis (4.6 kDa) isolated from goat showed inhibition zone against S. xylosus and E. coli (2 cm and 2.5 cm respectively), while bacteriocin of L. helveticus (kDa) showed inhibition zone against S. aureus (1.9 cm) and E. coli (1.8 cm) (Table 1B).

Bacteriocin activity among lactobacilli isolates

Bacteriocin of L. acidophilus of molecular weight (M.Wt=3.5 kDa) isolated from cow had no antibacterial effect on S. xylosus and Yersinia enterocolitica and bacteriocin of L. acidophilus of M.Wt 6.4 kDa isolated from cow had no effect against Yersinia enterocolitica . While bacteriocin of L. acidophilus of molecular weight 4 kDa isolated from cow inhibit growth of S. xylosus (1.9 cm) and Yersinia enterocolitica (2.5 cm). We also found that bacteriocin of L. pentosus of M.Wt (17 kDa) isolated from buffalo had no significant effect against S. uberis and Yersinia enterocolitica while bacteriocin of L. pentosus of molecular weight 9.8 kDa isolated from buffalo showed zone of inhibition against S. uberis (2.1 cm) and Yersinia enterocolitica (1.8 cm).

Effect of temperature and pH on bacteriocin activity

The effects of temperature and pH on bacteriocin activity among the examined lactobacilli are well illustrated in Tables 2-5 and Figure 2.

Figure 2: (A) Bacteriocins of L. acidophilus against Yersinia enterocolitica at 25°C and pH 7. (B) Bacteriocins of L. salivarius , L. pentosus and L. acidophilus against S. xylosus at 25°C and pH 7. (C) Bacteriocins of L. acidophilus against S. aureus at 25°C and pH 7. (D) Bacteriocins of L. salivarius and L. acidophilus against S. aureus at 40°C and pH 3. (E) Bacteriocins of L. fermentum against S. aureus at 25°C and pH 7. Bacteriocin of L. fermentum (10 kDa) has no inhibition effect on S. aureus at pH 7 and 25°C, but high inhibition at 80°C and pH 5 was 1.8 cm. Its inhibitory effect on E. coli at pH 7 and 25°C was 1.2 cm, which increased to 1.6 cm at 80°C and 1.5 cm at pH 5. The inhibition effect of S. xylosus at pH 7 and 25°C was 1.8 cm and no effect at 40°C. No inhibition effect against Yersinia enterocolitica at pH 7 and 25°C but at pH 5 and 100°C it was 1.4 cm and 1.5 cm respectively.

Table 2: Effect of temperature and pH on bacteriocin activity of L.fermentum (10 kDa) and L. salivarius (15 kDa) from cow isolates.

Table 3: Effect of temperature and pH on bacteriocin activity of L. acidophilus (8.3 kDa and 13.75 kDa) from buffalo and ewe isolates.

Table 4: Effect of temperature and pH on bacteriocin activity of L. delbrueckii subsp. bulgaricus (21 kDa) and L. rhamnosus (16.8 kDa) from Ewe isolates.

Table 5: Effect of temperature and pH on bacteriocin activity of L. brevis (4.6 kDa) from Goat isolates.

Data shown in Table 2 revealed that the bacteriocin of L. salivarius had inhibition effect on S. aureus (1.9 cm) at pH 7 and 25°C which increased to 2.6 cm at both pH 3 and 80°C. The bacteriocin had inhibition effect on S. uberis at pH 7 and 25°C reached to 2.3 cm. No increase of the zone of inhibition with increase of temperature but slightly increased at pH 3 (2.4 cm) was recorded. The bacteriocin has no effect on all pathogens at pH 10.

Table 3 illustrated that bacteriocin of L. acidophilus (8.3 kDa) had inhibition effect to S. aureus at pH 7 and 25°C (1.5 cm) and increased at 60°C to 1.8 cm and at pH 3 to 2.5 cm. Also in case of S. xylosus and S. uberis the inhibition zone increased from 1.3 cm and 1.2 cm at pH 7 and 25°C to 1.6 cm and 2.7 cm; 1.8 cm and 2.7 cm respectively at 60°C and pH 3. Inhibition zone against E. coli and Yersinia enterocolitica increased at 100°C and pH 3.

As shown in Table 4 showed, bacteriocin of L. acidophilus had inhibition effect to S. aureus at pH 7 and 25°C reached to 2 cm, increased at 40°C and pH 5 to 2.2 and 2.5 cm respectively. While no inhibition effect of the bacteriocin on S. xylosus at pH 7 and 25°C, but zone of inhibition reached to 1.6 cm at 60°C and 1.7 cm at pH, 8 and 10 each.

The inhibition effect of bacteriocin on S. uberis increased from 2 cm at pH 7 and 25°C to 2.6 cm at 100°C and 2.2 cm at pH 5. The inhibition effect of bacteriocin on E. coli and Yersinia enterocolitica increased at pH 5 but different in temperature, E. coli increased diameter of zone of inhibition at 40°C but Yersinia enterocolitica at 80°C.

The bacteriocin of L. delbrueckii subsp. bulgaricus had inhibition effect of S. aureus 1.7 cm at 25°C and pH 7 and no increase in inhibition zone by increase temperature but increased to 2.8 and 2.7 cm at pH 5 and 3 respectively. The bacteriocin has inhibition effect to S. xylosus and E. coli at 40°C (2 and 2.3 cm) and pH 5 (2.9 and 1.9 cm) respectively. And had inhibition effect to S. uberis and Yersinia enterocolitica at 80°C (2 and 1.7 cm) and pH 5 (2.9 and 1.8 cm) respectively (Table 4).

Table 4 concluded that the bacteriocin of L. rhamnosus had inhibition effect on S. aureus , S. xylosus , E. coli and Yersinia enterocolitica at pH 7 and 25°C with inhibition zone ranged from 1.6 to 1.9 cm. There was no increase of inhibition zone by increase temperature while it increased at pH 3. The bacteriocin of L. rhamnosus has no effect on pH 10.

Another set of data revealed that the bacteriocin of L. brevis had inhibition effect on S. aureus 1.8 cm at pH 7 and 25°C increased to 2.3 cm at 80°C and at pH 5 and 10 has inhibition zone 2.5 and 2.4 cm respectively, while no effect at pH 8. The bacteriocin has inhibition effect on S. xylosus 2 cm at pH 7 and 25°C then increased to 2.3 cm at 80°C and pH 5 but had no effect at pH 8. While no increased in zone of inhibition against S. uberis and E. coli by increased temperature and change in pH (Table 5).

Milk and milk products are usually associated with probiotic bacteria, which provide supplements in maintaining beneficial intestinal balance [22]. Lactobacilli (LAB) which can produce antibacterial agent against some major food spoilage and pathogenic bacteria are present in fresh cow milk, fermenting corn slurry and the feces of human neonates, pig and albino rat [23]. There were different bacilli species present in milk of cows, buffalo, ewe and goat which contain bacteriocin. These different bacteriocins have different impact on zone of inhibition which is dependent on temperature and pH.

The purified bacteriocin from different Lactobacillus isolates has wide range of variable molecular weight (3-30 kDa). Another finding reported the molecular weight of the bacteriocin from L. plantarum ST13BR as 10 kDa [24]. The bacteriocins of lactic acid bacteria belonging to class-I and II have molecular weight [25].

Various Lactobacillus species have been evaluated for the prevention or treatment of various infectious diseases and these were found to be safe [22]. L. acidophilus and L. rhamnosus showed high activity against the most predominant bacteria which were isolated from cases suffering from ovarian inactivity [26].

Bacteriocins are used to inhibit the growth of pathogenic organisms in foods [27]. All the lactic acid bacteria screened for bacteriocin production, Lactobacillus bulgaricus , L. lactis , L. acidophilus , Lactococcus lactis , Streptococcus thermophilus, S. cremoris , Pediococcus halophilus and P. cerevisiae produced bacteriocin activity between 4800 and 6000 Au/ml against Staphylococcus , Salmonella , Bacillus , Shigella and Pseudomonas species [28]. This property of inhibition made them potent bacteriocin producers.

The bacteriocin producing strain was isolated from bovine and ovine milk samples and the selected strain was identified. The susceptibilities of the examined Gram-positive (S. aureus , S. xylosus and S. uberis ) and Gram-negative bacteria (E. coli and Y. enterocolitica ) to growth inhibition by the bacteriocin of Lactobacillus species were recorded. Gilliland and Speck [29] reported that lactobacilli showed stronger antibacterial effect against Gram positive than Gram negative bacteria. It is clear that bacteriocin of the examined isolates shows antibacterial activity against all examined indicator bacteria except L. acidophilus bacteriocin (3.5 kDa) which had no antibacterial effect on S. xylosus and Yersinia enterocolitica isolates.

Interestingly, some bacteriocins from differet isolates have no effect on specific bacteria likewise bacteriocins of L. acidophilus (6.4 kDa) and L. brevis (4.6 kDa) had no effect against Yersinia enterocolitica whereas Bacteriocin of L. pentosus (17 kDa) had no effect against S. uberis and Yersinia enterocolitica. Also, bacteriocin of L. acidophilus (13.75 kDa) had no effect against S. xylosus , while bacteriocin of L. delbrueckii subsp. bulgaricus (21 kDa) had no effect against S. uberis and bacteriocin of L. fermentum (10 kDa) had no effect against S. aureus and Yersinia enterocolitica.

Similarly, bacteriocin from L. plantarum was found to be active against pathogenic bacteria including Cl. sporogenes , E. faecalis , E. coli and S. aureus [30,31]. Antibacterial activity of bacteriocin produced by isolated probiotics showed that, L. rhamnosus and L. plantarum had strong antibacterial effect against enteric bacterial pathogens [22].

L. Plantarum and L. rhamnosus from goat and L. plantarum from cow milk were more effect probiotic [22]. Lengkey and Adriani [32] showed the results about the sensitivity of the lactic acid bacteria on P. aeruginosa and S. aureus . Jin et al. [33] had earlier reported the inhibition of E. coli and Salmonella strains by Lactobacillus species from chicken intestine. Ehrmann et al. [34] reported the inhibition of fecal strains (E. coli, S. Enteritidis and S. Typhimurium ) by lactobacilli isolated from crops and intestine of ducks.

There is also reported the inhibition of food borne bacteria by bacteriocins from L. gasseri [35]. They observed that several strains of L. gasseri showed wide inhibitory activity against L. monocytogenes , Bacillus cereus , S. aureus , and E. coli . Recently Heredia-Castro et al. [36] recorded that Lactobacillus species from cheese were shown to produce bacteriocin-like substances active against S. aureus , L. innocua, E. coli and S. Typhimurium by using the disk diffusion method. Bacteriocin was active in a wide range of pH, but the maximum activity was observed at pH 3 and pH 5. Bacteriocins produced by L. plantarum and L. brevis retained their antimicrobial activity in an acidic pH range of 2.0 to 6.0, while inactivation occurred at pH 8.0 to 12.0 [37]. The present data revealed that most of the extracted bacteriocins of Lactobacillus species were stable in acidic as well as alkaline pH (3 to 10) except L. fermentum (10 kDa), L. rhamnosus (16.8 kDa) and L. salivarius (15 kDa) at pH 10.

The bacteriocins of L. acidophilus and L. bulgaricus were stable between pH 3 and pH 10 while L. helveticus was found to be sensitive to pH 10 [38]. Another study showed that bacteriocins of vaginal lactobacilli were stable at pH 4.5 to 7 but sensitive to pH 9 [39]. Bacteriocin-like substances produced by Lactobacillus strain showed potential for application as a food bio-preservative [36].

The effect of temperature on bacteriocin activity in terms of inhibition zones was study. Bacteriocins of all the selected Lactobacillus species were stable up to 100°C. It has been found to be thermostable in nature as it can withstand high temperature up to 100°C, although a partial loss in the activity was observed with a continuous increase in temperature. Thermo stability of bacteriocin at high temperature makes it possible to sterilize the food products even at room temperature. Earlier studies revealed that bacteriocins produced by L. para casei, L. lactis, L. plantarum and L. pentosus remained active after heating till 121°C for 20 min [40].

Bovine and ovine milk are conceder as a source of bacteriocin producers. Lactobacillus species could be of great interest in the production of bio preservatives for the food industries. The present study revealed that bacteriocins of Lactobacillus species from milk were stable at temperature up to 80°C and between pH 3 to 8 had strong antibacterial against many bacterial pathogens. Bacteriocin producers are recommended to food processing industries to enhance extension of shelf life of food products.

The authors declare that there is no conflict of interests.

The authors gratefully acknowledge Dr. Zafar Mahmood from Germany and Dr. Jens Hahne from England for their scientific input, experimental planning and revising the manuscript critically for important intellectual content.