Keywords

Probiotics; Antibacterial; Lactobacillus; Fermented milk; Intestinal pathogens

Introduction

Probiotics bacteria, have been recognized by FAO/WHO as live microorganism, which when ingestion in a particular number, confer a health benefit on the host [1,2]. Probiotics microorganisms have two main important sources, intestinal tract of human and fermented of dairy products [3]. These microorganisms may be included in foods or be marketed as supplement, in the form of tablets, capsules and by fermentation of dairy products and foods (non-dairy products) [4].

They are occurring in the intestine of healthy persons, micro flora of human gastrointestinal [5]. Various benefits have been described, particularly those pertaining to improvements in gastrointestinal diseases [6]. Many species of the genus of Lactobacillus are considered as a part of commensal intestinal flora of human and animal [7]. Certain strains of this genus can play as microbial barriers and they were able to produce inhibitory compounds such as lactic acid, hydrogen peroxide and proteinaceous compounds (e.g. bacteriocins) [8]. The main important groups of probiotic microorganisms are belonged to the Lactic Acid Bacteria (LAB) viz. Lactobacilli species, Enterococcus spp. and Bifidobacterium [9].

These groups of probiotics are consisted of a physiological and genetically diverse group of rod-shape, gram-positive, non-spore forming bacteria [10,11]. Catalase negative, microaerophilic to anaerobic, fastidious, strictly fermentative microorganisms and their main products as the results of fermentation path way is lactic acid [12,13]. Many studies have documented that Lactobacilli which isolated from dairy products have long history of safe use [14]. Several benefits have been found to be associated with the consumption of these bacteria including influencing immune system, also these probiotics microorganisms have ability to secret or produce antimicrobial or inhibitory substances such bacteriocins, and hydrogen peroxide. These metabolizes, have ability to inhibit both, gram positive and negative bacteria, with strong effect against gastric in the intestinal pathogens. This paper aimed to evaluate the antibacterial efficacy of probiotics (Lactobacilli spp.) against intestinal pathogenic bacteria (S. aureus, E. coli and S. typhi).

Materials and Methods

Collection of samples

Dairy products including fermented and raw samples were collected, about 12 samples, obtained from local markets in Khartoum state. Samples were preserved under aseptic conditions, and then transported to microbiological lab in an ice box to analyze in the same day.

Isolated and identification of Lactobacillus

From each sample 1 ml of fermented milk were diluted with 9 ml of sterile 0.1 peptone water (Difco) and homogenized serial dilution were prepared and 100 u from 10-10 dilutions of the samples were inoculated on plates of MRS agar medium, incubated at 37°C for 48-72 h under anaerobic condition using anaerobic jar. Plates containing 30-300 colonies were then enumerated. The colonies were characterized by their shape and color. Selected isolates also characterized for gram staining and biochemical tests.

Antibacterial activity of LAB to three pathogenic bacteria

Antibacterial activity was done on Muller Hinton Agar (MHA). Staphyloccus aureus, Escherichia coli, Salmonella typhi were sub-culture in MRS Broth. All bacterial cultures were diluted in 0.9% Nacl to 0.5% McFarland. A bacterial suspension of pathogenic bacteria was swabbed on (MHA) using sterile cotton. Suspensions of the LAB were dropped on the plates of MHA, the discs were placed on MHA, and then they were incubated at 370°C for 18-24 hr. The inhibition zone was measured.

Results and Discussion

Isolation of Lactobacillus bacteria

Nine samples were grown out 12 of samples shown in Table 1.

Table 1: Isolation of the Lactobacillus bacteria.

The identification

Form Table 2 represented nine rod-shape, gram-positive, nonspore, catalase negative, oxidase negative, non-motility bacteria were isolated from twelve samples. Nine of them were produced acid from glucose.

Table 2: Identification of lactic acid bacteria.

Antibacterial activity

Lactobacilli bacteria were cultured in pure MRS Broth, incubated for 72 hours, the antimicrobial was extracted from bacteria, by means of a centrifuge and then the filter paper was saturated with the extract and placed in the culture of pathogen bacteria (E. coli, S. aureus, S. typhi) and it was observed that there is a corona around that filter paper (inhibition zone) with different diameters shown in Table 3 (Figures 1-4).

Table 3: The diameter of inhibition zone of Lactobacilli against the pathogenic bacteria Lactobacillus spp. 1 and Lactobacillus spp. 2.

 

Figure 1: Culture of isolated LAB.

 

Figure 2: Inhibition zone of Lactobacillus against E. Coli.

 

Figure 3: Inhibition zone of Lactobacillus against Salmonella typhi.

 

Figure 4: Inhibition zone of Lactobacillus against Staphylococcus aureus.

On the assay of antimicrobial activity of LAB (Lactobacillus spp.) against three pathogenic bacteria showed that the (LAB, Lactobacillus isolates were capable to inhibit the pathogen. This results confirmed by Sridevi, et al., This indicate that LAB produced antibacterial metabolizes such as organic acid and bacteriocin during growth. E. coli was more sensitive bacteria; in contrast Salmonella typhi was have low activity.

Conclusion

Two LAB isolates were isolated from fermented milk. Of that, tow isolates (Lactobacillus) showed to inhibit to all three pathogenic bacteria, including E. coli, S. aureus and Salmonella typhi. The antimicrobial activity result obtained confirmed that the LAB (Lactobacillus) possesses an antibacterial activity. The antibacterial activity was more obvious against the gram negative bacteria E. coli then gram positive one, S. aureus.

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