Key Words

Bacteriocin, Lactobacillus, Antibacterial activity

Introduction

Bacteriocins are a kind of ribosomally synthesized antimicrobial peptides [3], which can kill or inhibit bacterial strains closely-related or non-related to produced bacteria, but will not harm the bacteria themselves by specific immunity proteins. Bacteriocins become one of the weapons against microorganisms due to the specific characteristics of large diversity of structure and function, natural resource, and being stable to heat [4]. Many recent studies have purified and identified bacteriocin for application in food technology, which aims to extend food preservation time [1] [2], treat pathogen disease and cancer therapy, and maintain human health. Therefore, bacteriocins may become a potential drug candidate for replacing antibiotics in order to treat multiple drugs resistant pathogens in the future. Bacteriocin is produce by Bifidobacterium, Enterococcus faecium, Rastonia solanacearum, non-pathogenic Escherichia coli, yeast like Saccharomyces boulardii and Lactic acid bacteria. Lactobacillus is widely used in the food industry as starter culture for fermentation [5]. Lactobacilli have been used since decades against infectious diseases and have been extensively studied for their ability to protect against pathogens. Lactobacillus bacteriocins are grouped as class-I bacteriocins (lantibiotics), class-II bacteriocins (heat-stable, nonlantibiotics), class-III bacteriocins (heat labile proteins with large molecular mass) and class-IV (hydrophobic and heatstable proteins, associated with lipids or carbohydrates). The lantibiotics in general have wider spectrum of activity than the non-lantibiotics [3].

Materials and Method

Sampling

The dairy products [6] [13] like cow milk, buffalo milk, goat milk, curd from Botad, homemade curd, meat product [10] like chicken, mutton, sea foods [12] like fish, black prawns, white prawns, alcoholic beverages like red wine, rose wine samples were collected in sterile screw cap tubes.(Table.1)

Table 1: Sampling sites for samples

Isolate Sites
C1 Curd from Pardi
PC1 Curd from Botad
M1 Buffalo milk from Pardi
CS1 Cow milk from Surat
G1 Goat milk from Olpad
OC1 Chicken from Olpad
OM1 Mutton from Olpad
F1 Fish from Olpad
BP1 Black prawns from Olpad
PP1 White prawns from Olpad
RS1 Rose wine from Sula
RD1 Red wine from Sula

Isolation of Lactobacillus

The collected samples were serially diluted (10-1 to 10ˉ 6 ) in sterile distilled water. The diluted samples were plated (0.1 ml suspension) onto de Man Rogosa Sharpe (MRS) agar plates [9] and incubated at 37ºC for 48 hours. In a total of 55 different colonies initially observed.

Phenotypic Identification

The Morphological and colony characteristics were studied using de Man Rogosa Sharpe (MRS) agar plates [9]. The physiological characteristics of all the obtained isolates were studied. The biochemical characteristics (Indole, Catalase, Voges-Proskauer, Methyl Red, Citrate, Hydrogen sulphide production, Nitrate reduction, Gelatin hydrolysis test, BileEsculin) and sugar fermentation tests were also carried out using standard reference Bergey’s Manual of Systematic Bacteriology [14].

Production of Bacteriocin

For the preparation of the inoculums, 10 ml of MRS broth was inoculated with 0.1 ml of freshly prepared culture of Lactobacillus spp. isolate and was incubated for 48 hours at 37ºC. From this pre-culture, 1 ml was added to 100 ml of MRS broth medium which after 48 hours of incubation at 37ºC, was used to inoculate the production media [16].

Antibacterial Activity against Indicator Bacteria

The recovered isolates were subjected for antibacterial activity by using agar well diffusion method [8] [10] [22]. The isolates and the indicator strains [Escherichia coli (MTCC No. 118), Salmonella typhi (MTCC No. 733), Staphylococcus aureus (MTCC No.737), and Bacillus cereus (MTCC No. 1305)] were grown in MRS broth and nutrient broth respectively [15]. The MRS broth were inoculated with 1 % (v/v) of the active culture of the recovered isolates and incubated at 37ºC for 48 hours. Cells were harvested by centrifugation at 6000 rpm for 15 minutes at 4 ºC and 50 µl supernatant were placed in 5mm diameter wells that had been cut in agar plates previously seeded with the indicator bacteria. The plates were incubated at 37ºC for 24 hours. After incubation, the diameter of zone of growth of inhibition was measured.

Results

Isolation of Lactobacillus spp. was carried out by using de Man Rogosa Sharpe (MRS) agar medium.

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Figure 1: de Man Rogosa Sharpe (MRS) agar medium

The total number of isolates were counted and further proceeded for their morphological and motility test. The colony characteristics of the obtained isolates were studied on de Man Rogosa Sharpe (MRS) agar plate, which gives white / creamy, circular, small / large colonies with entire margin after 48 hours of incubation (Fig 1). They were gram positive generally long rods sometimes they are short rods, coccoid and they were non motile. Following Table.2 shows result of various biochemical characteristics of the isolates.

Table 2: Biochemical characterization of the isolates.

Isolates Biochemical Tests Probable Identity
Tests Carbohydrates
Indole Catalase Methyl Red V-P Test H₂S Production Nitrate Reduction Gelatin Hydrolysis Bile-Esculin Arabinose Cellobiose Lactose Mannitol Melibiose Salicin Sorbitol Sucrose Raffinose Trehalose Xylose
C1 + + + + + + + L. lactis
C2 + + + + + + L. fermentum
C3 + + + + + + + + + L. plantarum
C4 + + + + + + + + + L. plantarum
C5 + + + + + L. sakei
C6 + + + + + + L. buchneri
C7 + + + + + + + + + + L. plantarum
C8 + + + + + + L. buchneri
C9 + + + + + + L. fermentum
M1 + + + + + + + L. lactis
M2 + + + + + L. sakei
M3 + + + + + + + + + + L. plantarum
M4 + + + + + + + + + + L. plantarum
PC1 + + + + + + L. fermentum
PC2 + + + + + + + + + L. plantarum
PC3 + + + + + + L. brevis
PC4 + + + + + + + + + + L. plantarum
PC5 + + + + + + L. buchneri
PC6 + + + + + L. sakei
PC7 + + + + + + + L. lactis
PC8 + + + + + L. sakei
G1 + + + + + + L. fermentum
G2 + + + + + + L. fermentum
G3 + + + + + + L. brevis
OM1 + + + + + L. alimentarius
OM2 + + + + L. sakei
OM3 + + + L. curvatus
OM4 + + + + + L. alimentarius
OC1 + + + + + + L. fermentum
OC2 + + + + + + L. fermentum
OC3 + + + + + + L. fermentum
OC4 + + + + + + + + + L. plantarum
CS1 + + + + + + + L. lactis
CS2 + + + + + + L. fermentum
CS3 + + + + + + + + + L. plantarum
CS4 + + + + + + + L. casei
CS5 + + + + + + + L. brevis
F1 + + + + + + L. brevis
F2 + + + + + + L. fermentum
F3 + + + + + + + + + L. casei
F4 + + + + + + L. fermentum
F5 + + + + + + + + + L. plantarum
F6 + + + + + L. alimentarius
BP1 + + L. curvatus
BP2 + + + + + + L. alimentarius
BP3 + + + + + + L. fermentum
BP4 + + + L. curvatus
BP5 + + + + + + L. fermentum
PP1 + + + + + + + L. lactis
PP2 + + + + + L. farciminis
PP3 + + L. curvatus
PP4 + + + + + + + + L. plantarum
RS1 + + + + + + L. buchneri
RD1 + + + + + + L. fermentum
RD2 + + + + + + L. fermentum

‘+’: Positive, ‘-‘: Negative

By studying the morphology and biochemical characteristics, we found various species of Lactobacillus such as Lactobacillus fermentum, Lactobacillus lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus alimentarius, Lactobacillus buchneri, Lactobacillus sakei, Lactobacillus curvatus, and Lactobacillus farciminis. The total number of isolates and its types are shown in fig 2.

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Figure 2: Isolated species of Lactobacillus

Bacteriocin Assay

In total of 55 isolates, 24 isolates were able to produce bacteriocin. (6 from homemade curd, 2 from buffalo milk, 8 from town curd, 1 from cow milk, 1 from white prawns, 2 from black prawns , 2 from chicken and 2 from mutton).

Agar well Diffusion Assay

Bacteriocins obtained from the isolates showed inhibitory activity against Escherichia coli (MTCC No.118), Staphylococcus aureus (MTCC No.737) and Bacillus cereus (MTCC No.1305). Salmonella typhi (MTCC No.733) was resistant to bacteriocin producers. PC3, PC5, OC4, and OM4 showed very strong inhibition (Fig 3) against Bacillus cereus (MTCC No.1305) with zone of inhibition 20mm, 22mm, 19mm, and 18mm (Table No.3).

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Figure 3: Antibacterial activity of Bacteriocin

Table 3: Inhibition of indicator bacteria by bacteriocin produced by isolates

ISOLATE NO. INDICATOR STRAIN
Bacillus cereus (MTCC NO.1305) Escherichia coli (MTCC NO. 118) Staphylococcus aureus ( MTCC NO.737) Salmonella typhi (MTCC NO.733)
C1 15mm 12mm
C2 17mm 9mm
C3 12mm 15mm
C4 16mm 12mm
C5 19mm
C9 12mm
M1 15mm
M2 12mm 7mm 10mm
PC1 17mm 5mm
PC2 6mm
PC3 20mm 8mm
PC4 17mm
PC5 22mm
PC6 12mm
PC7 15mm 5mm
PC8 13mm 6mm
OM1 15mm
OM4 17mm 17mm
OC3 12mm
OC4 15mm 15mm
CS5 12mm
BP4 12mm
BP5 12mm
PP1 15mm

‘-’: No zone of inhibition

C6,C7, C8, M3, M4, G1, G2, G3, OM2, OM3, OC1, OC2, CS1, CS2, CS3, CS4, F1, F2, F3, F4, F5, F6, BP1, BP2, BP3, PP1, PP2, PP3, PP4, PP5, RS1 and RD2 were not able to produce inhibitory effect against indicator bacteria.

Discussion

The study highlights the screening, production and antibacterial activity of bacteriocin from Lactobacillus spp. A total of 55 isolates were obtained from dairy products, meat, fish and wine samples. Isolates were confirmed as Lactobacillus spp. based on their morphological and biochemical characteristics. Kandler and Weiss (1986) have classified Lactobacillus isolates according to their morphology, physiology and biochemical test. Lactobacillus plantarum and Lactobacillus fermentum found in almost all samples. Singh and Rakesh Roshan Sharma (2009) stated that Lactobacillus fermentum and Lactobacillus casei were found in cow milk. Same results were found in our study. Among 55 isolates 24 isolates were able to produce bacteriocin. Isolates were show antibacterial activity against Escherichia coli (MTCC No.118), Staphylococcus aureus (MTCC No.737), and Bacillus cereus (MTCC No.1305) whereas; Salmonella typhi (MTCC No.733) was resistant to bacteriocin. Daeschel and Mc Kenney (1990) found the highest inhibitory activity by Lactobacillus plantarum. In red wine sample same result was not found in our study.

Conclusion

The study revealed that bacteriocin from Lactobacillus spp. isolated from different samples possesses a wide spectrum of inhibitory activity against Staphylococcus aureus (MTCC No.737), Bacillus cereus (MTCC No.1305) and Escherichia coli (MTCC No.118). Therefore, it has a potential for application as a biopreservative in different food products as such or in combination with other preservation methods.