**3.2. Screening of** *Lactobacilli* **strains for antimicrobial activity and bacteriocin production**

Eighty four lactobacilli strains previously isolated from two species of Sturgeon fish identied and their cell free supernatant extracts were assayed for antimicrobial activity and


**3. Results** 

**3.1. Isolation of lactobacilli** 

MRS 4.2: deMan, Rogosa and Sharp

**production** 

sturgeon and beluga in different media

visualized by an inhibition zone around the active protein band [39].

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

Intestinal content of 22 fish were analysed for the presence of lactobacilli. To determine the most appropriate medium for isolating lactobacilli from fish intestines, two media (MRS agar, LAMVAB) were used. LAMVAB was highly selective to quantify lactobacilli, as 99% of 143 randomly picked colonies and purified isolates were identified as *Lactobacillus* spp. and confirmed according to [12] (Table 1). Counts of intestinal lactobacilli for Persian sturgeon and beluga were detected at the range of approximately 105.3 to 106.4 cfu/g, respectively. The physiological and biochemical characterization of *Lactobacillus* isolates and the presumptive *Lactobacillus* species found in two fish species are shown in Table 2. From 84 isolates, 2 metabolic groups of *Lactobacillus* were recovered: facultative and obligate heterofermentatives. *L. sakei* and *L. plantarum* were the most often found isolates (Table 2). MRS 4.2 was suitable to quantify lactobacilli. As 30 randomly picked colonies on the highest dilution were identified as lactobacilli and coccoid forms were not found. Means of counts of 90 samples were not statistically different to LAMVAB counts in the Student's t-test (P=0.29) and were correlated with LAMVAB counts (r = 0.85; P<0.001). The correlation of counts on MRS 4.2 with those on LAMVAB and the absence of coccoids suggests that lactobacilli were the most important acidophilic lactic acid bacteria in the samples analysed. Facultative anaerobic flora recovered

in CAB medium provided the highest counts in the samples analysed (Table 1).

*Acipenser persicus* 12 7.84 5.32 4.85 *Huso huso* 10 8.21 6.45 5.64

Fish species No. CAB (cfu/g) LAMVAB (cfu/g) MRS 4.2 (cfu/g)

CAB: Columbia blood agar; LAMVAB: *Lactobacillus* spp. Anaerobic MRS with Vancomycin and Bromocresol green;

**Table 1.** Average bacterial counts of intestinal bacteria (Log cfu/g of intestinal content) for Persian

**3.2. Screening of** *Lactobacilli* **strains for antimicrobial activity and bacteriocin** 

Eighty four lactobacilli strains previously isolated from two species of Sturgeon fish identied and their cell free supernatant extracts were assayed for antimicrobial activity and † +: 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-Sorbose, Dulcitol, Inulin, Glycogen, D-Fucose, L-Fucose, L-Arabitol. ND: Not data

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

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

method and well diffusion method. In each instance, diameters of inhibition were quantied. Fifteen strains (18%) exhibited inhibitory activity against both indicator organisms. Consequently, all candidate isolates (Inhibition zone> 8mm) subjected to different tests such as growth at different temperatures, pH, salt content, antibiotic resistance, etc. Based on the result of aforementioned tests, two strains *Lactobacillus* casei AP8 and *Lactobacillus plantarum* H5, isolated from Persian sturgeon and beluga respectively, were chosen as active strains and were subjected to further examinations.

Selection of *Lactobacillus* Species from Intestinal Microbiota of Fish for Their Potential Use as Biopreservatives 413

**Indicator organism Medium\* Temp. [°C] Bac AP8 Bac H5** 

*Aeromonas hydrophilus* MJ 1120 **BHI 37 ++ 0**  *Aeromonas hydrophilus* MJ 1240 **BHI 37 +++ +**  *Aeromonas salmonicida* CC 1546 **BHI 37 + +**  *Aeromonas salmonicida* RT 7895 **BHI 37 ++ +**  *Brochothrix thermosphacta* RF 35 **BHI 37 ++ +**  *Escherichia coli* ATCC 25922 **BHI 37 ++ 0**  *Escherichia coli* PTCC 1325 **BHI 37 ++ ++**  *Photobacterium damselae ssp. Piscida* **BHI 37 0 0**  *Pseudomonas aeruginosa* PTCC 1310 **BHI 37 ++ +**  *Pseudomonas uorescens* HFC 1236 **BHI 37 ++ 0**  *Salmonella enteritidis* ATCC 13076 **BHI 37 ++ ++**  *Salmonella spp* SM 162 **BHI 37 +++ ++**  *Vibrio anguillarum* MI12 **BHI 37 ++ +**  *Vibrio parahaemolyticus* MI 23 **BHI 37 +++ 0**  *Vibrio parahaemolyticus MI 56* **BHI 37 +++ +** 

*Bacillus cereus* ATCC 9634 **BHI 37 +++ +++**  *Bacillus coagulans* **BHI 37 +++ ++**  *Bacillus licheniformis* PTCC 1331 **BHI 37 ++ 0**  *Bacillus subtilis* ATCC 9372 **BHI 37 +++ +**  *Clostridium perfringens* ATCC 3624 **RCM 37 ++ +**  *Clostridium sporogenes* PTCC 1265 **RCM 37 ++ +**  *Lactobacillus acidophilus* ATCC 4356 **MRS 30 ++ +**  *Lactobacillus alimentarius* AP 10 **MRS 30 + ++**  *Lactobacillus brevis* H56 **MRS 30 ++ ++**  *Lactobacillus brevis* AP 83 **MRS 30 ++ ++**  *Lactobacillus casei* PTCC 1608 **MRS 30 0 ++**  *Lactobacillus casei* RN 78 **MRS 30 0 0**  *Lactobacillus casei* LB 10 **MRS 30 0 +**  *Lactobacillus casei* LB 46 **MRS 30 0 +**  *Lactobacillus plantarum* PTCC 1050 **MRS 30 0 0**  *Lactobacillus plantarum* AP 76 **MRS 30 + 0**  *Lactobacillus plantarum* H12 **MRS 30 + 0**  *Lactobacillus sakei* AP 43 **MRS 30 0 +**  *Lactobacillus sakei* **MRS 30 0 0**  *Lactococcous sp* **MRS 30 + 0**  *Lactobacillus curvatus* **MRS 30 0 +**  *Listeria innocua AN 15* **BHI 37 ++ ++**  *Listeria monocytogenes* ATCC 7644 **BHI 37 +++ +++**  *Listeria monocytogenes* PTCC 1163 **BHI 37 ++ ++**  *Listeria monocytogenes* PTCC1297 **BHI 37 ++ ++**  *Staphylococcus aureus* ATCC 25923 **BHI 37 +++ +**  *Staphylococcus aureus* PTCC 1112 **BHI 37 +++ +** 

\* BHI: brain hearth infusion, MRS: de Man-Rogosa-Sharpe agar and RCM: reinforced clostridial medium. 0 no zone of inhibition; +, 1 mm<zone<5 mm; ++, 5 mm<zone<8 mm; +++, zone>8 mm.; PTCC: Persian Type Culture Collection;

**Table 4.** Antimicrobial activity of potential bacteriocin producing strain *L. casei* AP8 *and L. plantarum* 

**Gram Negative Group**

**Gram Positive Group**

ATCC: American Type Culture Collection.

H5 as examined with selected bacterial indicator strains.


\* = Presence of lactobacilli. \*\* = High number of lactobacilli presence

**Table 3.** *Lactobacillus* species isolated from the intestines of sturgeon fish
