**2.7. Growth dynamics and antimicrobial compounds production**

408 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

and Hammes and Vogel [32].

**2.4. Statistical analysis** 

p<0.05 was used.

surrounding the disks.

**2.6. Characterization of the inhibitory effect** 

manufacturer's instructions (Biomerieux, Marcy l' Etoile, France). The APILAB PLUS database identification software (bioMe´rieux, France) was used to interpret the results. Identification was undertaken according to the method described by Kandler and Weiss [12]

Statistical analysis using Student's t-test (SPSS, Version 11.0) was performed to find significant difference on lactobacilli count between LAMVAB and MRS 4.2. Pearson's correlation coefficient was used to investigate the correlation of lactobacilli count between LAMVAB and MRS 4.2 (SPSS Inc., Version 11.0, Chicago, USA). A significance level of

In a first test series, the ability of each of the *Lactobacillus* isolates to exert an antibacterial effect against *Listeria monocytogenes* ATCC 19115 and *Salmonella* Typhimurium PTCC 1186 were examined by using three methods: the spot-on-lawn method, standardized agar disk diffusion method and the well diffusion method as described by Schillinger and Lucke [33], Benkerroum et al. [34] and Tagg & Mc Given [35]. Throughout, cell-free supernatants (CFS) of strains were obtained by centrifugation at 10,000 ×g for 20 min and then adjusted to pH 6.5 by applying NaOH (to exclude the effect of organic acid) before sterilization by filter (0.2 µm, Sigma, UK). Based on the screening tests, the inhibitory spectrum of potential bacteriocin-producing isolates was assessed against 42 indicator strains using a standardized agar disk diffusion test. The strains were kept frozen in 20% (v/v) glycerol at - 20°C. For this purpose, an aliquot of 20 ml CFS was applied on disks (6 mm) and set on agar plates previously inoculated with each individual indicator strain suspension, which corresponded to a 105 CFU/ml. Plates were incubated 24 h at optimum temperatures of the test organism. Antimicrobial activity was detected as a translucent halo in the bacterial lawn

In order to determine the biological nature of the antimicrobial activity of bacteria, CFS (pH 6.0) of 24-h lactobacilli cultures of two selected isolates (*Lactobacillus casei* AP8 & *Lactobacillus plantarum* H5) incubated at 30°C, were tested for their sensitivity to the proteolytic enzymes. One ml of CFS was treated for 2 h with 1 mg ml-1 final concentration of the following enzymes: papain, trypsin, proteinase K, pronase E and α-amylase (Sigma, London). To clarify whether the antimicrobial activity detected derives from the production of hydrogen peroxide, 2600 IU/ml of catalase (Sigma, London) were added to 1 ml portions of extracellular extracts of LAB exhibiting antimicrobial activity and incubated for 24 h at ambient temperature. Chemicals were added to the CFS and the samples incubated for 5 h before being tested for antimicrobial activity. To determine the sensitivity of potential bacteriocin activities to the temperatures, samples of CFS were incubated under defined conditions. The effect of pH on bacteriocin

**2.5. Screening of** *Lactobacillus* **strains for their inhibitory potential** 

The time course of inhibitory substance production was performed by inoculating 10 mL of an overnight culture of selected *Lactobacillus* isolates into 100 mL of MRS broth followed by incubation at 30°C. Cells were subsequently removed by centrifugation at 10,000 ×g for 20 min. At appropriate intervals, changes in pH and optical density (600 nm) of the cultures were measured to monitor bacterial growth using a spectrophotometer (Hitachi U 1100, Tokyo, Japan). Antibacterial activity was evaluated every hour by using serial twofold dilutions of each culture used as a neutralized cell-free supernatant (CFS) tested against *L. monocytogenes* ATCC 19115 based on the agar disk diffusion plate bioassay. In a separate experiment, the inhibitory effect of CFSs of lactobacilli strains on target cells in liquid medium was also examined against *L. monocytogenes* ATCC 19115 as indicator strain. For this purpose, 20 mL of each filter-sterilized bacteriocin-containing cell-free supernatant were added to a 100 mL culture of the indicator organism at early exponential phase (4 h old). These experiments were also repeated with stationary-phase cells. The optical density at 600 nm and viable cell count were determined every hour during an observation period of 20 h. Indicator cells without CFSs were used as control.

### **2.8. Adsorption of bacteriocin to producer cells**

Bacteriocin-producing cells were cultured for 18 h at 30 °C. The pH of the cultures was adjusted to 6.0 with 1 M NaOH to allow maximal adsorption of the bacteriocin to the producer cells, according to the method described by Yang et al. [36]. The cells were then harvested (10,000 ×g 20 min, 4 °C) and washed with sterile 0.1 M phosphate buffer (pH 6.5). The pellet was re-suspended in 10 ml of 100 mM NaCl (pH 2.0) and stirred slowly for 1 h at 4 °C. The suspension was then centrifuged (10,000 ×g 20 min, 4 °C), the CFS was neutralized to pH 7.0 with sterile 1 M NaOH followed by testing the bacteriocin activity as described above.

### **2.9. Partial purication and characterization of the bacteriocin**

Bacteriocin producer strains were grown in MRS broth, and incubated without agitation for 18 h at 30°C. The cells were harvested (10,000 ×g, 20 min, 4 °C) and the bacteriocin precipitated from the CFS with 60% saturated ammonium sulphate [45]. The precipitate in the pellet and oating on the surface were collected and re-suspended in one-tenth volume 25 mM ammonium acetate buffer (pH 6.5). The sample was stored at -20 °C for one week and activity tests were performed as described above. For the determination of the molecular size of the bacteriocins, precipitated peptides re-suspended in 25 mM ammonium acetate buffer (pH 6.5) were separated by Tricine-SDS-PAGE, according to Schägger and Von Jagow [38]. Low molecular weight markers, ranging from 2.5 to 45 kDa (Pharmacia, Sweden) were used. One half of the gel containing the molecular marker was fixed for 20 min in 5% (v/v) formaldehyde, then rinsed with water and stained with Coomassie Brilliant Blue R250 (Bio-Rad) overnight. The other half of the gel (not stained and extensively pre-washed with sterile distilled water) was overlaid with a culture of 106 cfu/ml *L. monocytogenes* ATCC 19115 embedded in BHI agar. The position of the active bacteriocin was visualized by an inhibition zone around the active protein band [39].

Selection of *Lactobacillus* Species from Intestinal Microbiota of Fish for Their Potential Use as Biopreservatives 411

*L. alimentarius*

*L. brevis*

*L. casei* 

*L. oris* 

*2* 

5

7

10

*L. coryneformis*

12

Diaminopimelic acid ND + ND ND ND ND *ND*  CO2 from glucose - - - - + - *+* NH3 from arginine - - - - + - *+* 10°C ++ + + + + *+* 15°C ++ + + + + *+* 45C - - - 2 - - *-* Glycerol - + - 1 - + *-* L-Arabinose + + - 2 2 - *+* Ribose + - - + + + *+* D-Xylose 26 - - - - - *+* Galactose 29 - - - - + *-* Rhamnose - - + - 2 + *-* Inositol - + - - - + *+* Mannitol - + 5 - + + *-* Sorbitol - + - - - + *-* 1-Methyl-D-mannoside - + - - - - *+* 1-Methyl-D-glucoside - + - 7 + - *+* N-Acetyl glucosamine 28 + + + + + *+* Amygdaline 10 + - + - + *+* Arbutine 1 + - + - + *+* Esculine + + + + 1 + *+* Salicin + + - + - + *+* Cellobiose 27 + - + - + *+* Maltose 19 + - + + + *+* Lactose 26 + - + - + *+* Melibiose + + + 2 + - *+* Sucrose + + + 8 + + *+* Trehalose + + - + - + *-* Melezitose - + - - + + *+* D-Raffinose 29 - - 2 + - *-* Starch - - - - - + *-* Xylitol - + 3 - - - *+* 2-Gentiobiose + + - + - + *+* D-Turanose - - - - + - *-* D-Tagatose 1 - - + - + *-* D-Arabitol - + 5 - - - *+* Gluconate + - - + + + *+* 2-keto-gluconate - - 1 2 - - *+* 5-keto-gluconate - - - 1 - + *+ Lactic acid configuration DL DL DL DL DL DL D* † +: Positive reaction of all the isolates. Numbers are the positive isolates. All isolates fermented D-Glucose, D-Fructose, D-Mannose, however they didn't ferment erythrol, D-Arabinose, L-Xylose, Adonitol, 2-Methyl-xyloside, L-

Presumptive *Lactobacillus* species No. of isolates

*L. sakei*

*L. plantarum*

18

Sorbose, Dulcitol, Inulin, Glycogen, D-Fucose, L-Fucose, L-Arabitol. ND: Not data

sturgeon and beluga

**Table 2.** Biochemical characteristics of *Lactobacillus* species isolated from the intestines of Persian

bacteriocin production against *Listeria monocytogenes* ATCC 19115 and *Salmonella*  Typhimurium PTCC 1186 by using spot-on-lawn method, standardized agar disk diffusion

30
