**3.3. Inhibitory spectrum of bacteriocin**

As the results in screening test showed that greater inhibition was observed by agar disk diffusion tests of cell-free supernatant extracts, so this method was selected as the best technique for examining the antibacterial activity of *L. casei* AP8 and *L. plantarum* H5 CFSs against forty two Gram-positive and Gram-negative bacteria. The CFS preparations from both strains showed a broad inhibitory spectrum against a wide range of LAB of different species and some food-borne pathogens and spoilage bacteria including *Listeria innocua*, *L. monocytogenes*, *Staphylococcus aureus*, *Aeromonas hydrophila*, *Aeromonas salmonicida*, *Bacillus cereus*, *Bacillus pumilus*, *Bacillus subtilis*, *Brochotrix thermosphacta*, Gram-negative *E. coli*, *Salmonella* and *Pseudomonas*, *Clostridium perfringens* and *Vibrio parahaemolyticus* (Table 4). Result showed that the Gram-positive bacteria tested were more sensitive to the bacteriocin produced by the isolates than Gram-negative bacteria. The largest spectrum of inhibition was showed by *L. casei* AP8 bacteriocin, which inhibited 33 out of 42 indicator strains.

### **3.4. Characterization of inhibitory effect**

Table 5 and table 6 depict the stability of inhibitory substances at different physic-chemical conditions. To determine the biological nature of the antimicrobial activity of bacteria, CFSs were tested for their sensitivity to the proteolytic enzymes. Antimicrobial activities exhibited by *L. casei* AP8 and *L. plantarum* H5 were sensitive to proteolytic enzymes since proteolytic, but not lipolytic or glycolytic enzymes, completely inactivated the antimicrobial effect of both cell-free supernatants, conrming the proteinaceous nature of the inhibitors (Table 3). The effect of several chemicals on the antimicrobial activity was also evaluated. Interestingly, the cell-free extracts remained active after treatment with chemicals such as catalase, SDS, Triton X-100, Tween 20, Tween 80 and EDTA after 5 h of exposure (Table 2). Enhancing the antimicrobial activity in case of *L. casei* AP8 bacteriocin was observed after treating by EDTA and SDS against *L. monocytogenes* ATCC 19115. The stability study of

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

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

**3.3. Inhibitory spectrum of bacteriocin** 

**3.4. Characterization of inhibitory effect** 

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

Presumptive *Lactobacillus* species

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,

*Acipenser persicus* \*\* \*\* \* \*\* - \*\* *\* Huso huso \*\* \* - \* \*\* \* \** 

As the results in screening test showed that greater inhibition was observed by agar disk diffusion tests of cell-free supernatant extracts, so this method was selected as the best technique for examining the antibacterial activity of *L. casei* AP8 and *L. plantarum* H5 CFSs against forty two Gram-positive and Gram-negative bacteria. The CFS preparations from both strains showed a broad inhibitory spectrum against a wide range of LAB of different species and some food-borne pathogens and spoilage bacteria including *Listeria innocua*, *L. monocytogenes*, *Staphylococcus aureus*, *Aeromonas hydrophila*, *Aeromonas salmonicida*, *Bacillus cereus*, *Bacillus pumilus*, *Bacillus subtilis*, *Brochotrix thermosphacta*, Gram-negative *E. coli*, *Salmonella* and *Pseudomonas*, *Clostridium perfringens* and *Vibrio parahaemolyticus* (Table 4). Result showed that the Gram-positive bacteria tested were more sensitive to the bacteriocin produced by the isolates than Gram-negative bacteria. The largest spectrum of inhibition was showed by *L. casei* AP8 bacteriocin, which inhibited 33 out of 42 indicator strains.

Table 5 and table 6 depict the stability of inhibitory substances at different physic-chemical conditions. To determine the biological nature of the antimicrobial activity of bacteria, CFSs were tested for their sensitivity to the proteolytic enzymes. Antimicrobial activities exhibited by *L. casei* AP8 and *L. plantarum* H5 were sensitive to proteolytic enzymes since proteolytic, but not lipolytic or glycolytic enzymes, completely inactivated the antimicrobial effect of both cell-free supernatants, conrming the proteinaceous nature of the inhibitors (Table 3). The effect of several chemicals on the antimicrobial activity was also evaluated. Interestingly, the cell-free extracts remained active after treatment with chemicals such as catalase, SDS, Triton X-100, Tween 20, Tween 80 and EDTA after 5 h of exposure (Table 2). Enhancing the antimicrobial activity in case of *L. casei* AP8 bacteriocin was observed after treating by EDTA and SDS against *L. monocytogenes* ATCC 19115. The stability study of

*L. sakei L. plantarum L. coryneformis L. alimentarius L. brevis L. casei L. oris* 

were chosen as active strains and were subjected to further examinations.


\* 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; ATCC: American Type Culture Collection.

**Table 4.** Antimicrobial activity of potential bacteriocin producing strain *L. casei* AP8 *and L. plantarum*  H5 as examined with selected bacterial indicator strains.

inhibitory compounds of *L. casei* AP8 and *L. plantarum* H5 in different conditions indicated the high resistance of these agents. The antimicrobial compounds were able to resist most of these factors to which it was exposed even during prolong incubation period (Table 6). Cell free extracts prepared from both the isolates are found to be thermo-stable. When *L. casei*  AP8 bacteriocin was heated at 40-100° C for 30 min, it retained inhibitory activity against *L. monocytogenes* ATCC 19115. However, a loss in activity in the ranges of 35% was observed when heated at 120°C for 15 min (Table 6). The Antilisterial activity of *L. plantarum* H5 bacteriocin was resistant to heat treatments of 40-100°C for 30 min and remained constant after heating at 121ºC for 15 min. Both investigated bacteriocins were most stable at 4°C and - 20°C and able to retain their antilisterial activity for 30 days without any decrease. *L. casei*  AP8 bacteriocin was active in a wide range of pH, as full activity was retained at pH values between 3 and 10. *L. plantarum* H5 bacteriocin remained stable after incubation for 2 h at pH values between 2.0 - 12.0.

Selection of *Lactobacillus* Species from Intestinal Microbiota of Fish for Their Potential Use as Biopreservatives 415

+ + + [-35%] -

> - + - -

**Table 6.** Effect of cold storage, different temperatures and pH on inhibitory activity against *Listeria* 

Figure 1 shows the growth and bacteriocin production curves of *L. casei* AP8 and *L. plantarum H5* cultured at 30°C. For *Lactobacillus* casei AP 8 cell growth reached the stationary phase at 12 h of cultivation. Kinetics of bacteriocin production showed that its synthesis and/or secretion started at 4 h growth in the exponential phase of growth and maximum activity was observed at the early stationary phase of growth (1800 AU ml-1) and had stabled for 6 h before the bacteriocin activity decreased (Figure 1). The pH values decreased from 6.5 to 3.7 at the end of incubation. For *L. plantarum* H5, bacteriocin activity was detectable in the culture supernatant after 5hr when an absorbance of 0.55 at 600 nm of the culture broth. Production of bacteriocin increased throughout logarithmic growth. In the stationary phase, *L. plantarum* H5 showed maximum bacteriocin activity (3400 AU/mL) and stabilized for 2 hr. But since then, bacteriocin activity declined gradually and stabilized at 1600 AU/ml during the following 4 h. In the stationary phase, extracellular pH was maintained, however, bactericidal activity decreased, excluding a possibility of lactic acid as a bactericidal

**Figure 1.** Antimicrobial activity [bars] against *L. monocytogenes* ATCC19115 of *L. casei* AP8 [A] and *L. plantarum* H5 [B] observed during growth in MRS medium [●] and expressed in AU/ml. Results are

Residual antimicrobial activity

+ + + +

+ + + +

*L. casei* AP8 *L. plantarum* H5

Treatment (Storage,

4 ºC, -20ºC/ 30 d 40-100 ºC/30 min 121 ºC/10 min 121 ºC/15 min

pH= range 3-10

No inhibition= **-**; inhibition= **+**

*monocytogenes* ATCC 19115. For details see text.

**3.5. Growth and bacteriocin production** 

represent the mean of three independent experiments.

pH= 2

pH= 11 pH= 12

mechanism.

Temperature and pH stability)


**Table 5.** Effect of enzymes and chemicals on the antimicrobial activity of two selected strains *L. casei*  AP8 and *L. plantarum* H5. For details see text



No inhibition= **-**; inhibition= **+**

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

values between 2.0 - 12.0.

**Enzymes**  Trypsin Papain Proteinase K Pronase E α- amylase Catalase

**Organic solvents** 

Butanol Ethanol Methanol Ethyl ether EDTA Sodium deoxycholate Sulphobetaine 14

SDS Tween 20 Tween 80

inhibitory compounds of *L. casei* AP8 and *L. plantarum* H5 in different conditions indicated the high resistance of these agents. The antimicrobial compounds were able to resist most of these factors to which it was exposed even during prolong incubation period (Table 6). Cell free extracts prepared from both the isolates are found to be thermo-stable. When *L. casei*  AP8 bacteriocin was heated at 40-100° C for 30 min, it retained inhibitory activity against *L. monocytogenes* ATCC 19115. However, a loss in activity in the ranges of 35% was observed when heated at 120°C for 15 min (Table 6). The Antilisterial activity of *L. plantarum* H5 bacteriocin was resistant to heat treatments of 40-100°C for 30 min and remained constant after heating at 121ºC for 15 min. Both investigated bacteriocins were most stable at 4°C and - 20°C and able to retain their antilisterial activity for 30 days without any decrease. *L. casei*  AP8 bacteriocin was active in a wide range of pH, as full activity was retained at pH values between 3 and 10. *L. plantarum* H5 bacteriocin remained stable after incubation for 2 h at pH

Treatment Concentration %Residual antimicrobial activity

1 mg/ml-1 1 mg/ml-1 1 mg/ml-1 1 mg/ml-1 1 mg/ml-1 1mg/ml-1

10% [v v] 10% [v v] 10% [v v] 10% [v v] 5 mmol l-1 1mg ml-1 1mg ml-1 1% [w v] 1% [v v] 1% [v v]

AP8 and *L. plantarum* H5. For details see text

*L. casei* AP8 *L. plantarum* H5

**Table 5.** Effect of enzymes and chemicals on the antimicrobial activity of two selected strains *L. casei* 

**Table 6.** Effect of cold storage, different temperatures and pH on inhibitory activity against *Listeria monocytogenes* ATCC 19115. For details see text.

### **3.5. Growth and bacteriocin production**

Figure 1 shows the growth and bacteriocin production curves of *L. casei* AP8 and *L. plantarum H5* cultured at 30°C. For *Lactobacillus* casei AP 8 cell growth reached the stationary phase at 12 h of cultivation. Kinetics of bacteriocin production showed that its synthesis and/or secretion started at 4 h growth in the exponential phase of growth and maximum activity was observed at the early stationary phase of growth (1800 AU ml-1) and had stabled for 6 h before the bacteriocin activity decreased (Figure 1). The pH values decreased from 6.5 to 3.7 at the end of incubation. For *L. plantarum* H5, bacteriocin activity was detectable in the culture supernatant after 5hr when an absorbance of 0.55 at 600 nm of the culture broth. Production of bacteriocin increased throughout logarithmic growth. In the stationary phase, *L. plantarum* H5 showed maximum bacteriocin activity (3400 AU/mL) and stabilized for 2 hr. But since then, bacteriocin activity declined gradually and stabilized at 1600 AU/ml during the following 4 h. In the stationary phase, extracellular pH was maintained, however, bactericidal activity decreased, excluding a possibility of lactic acid as a bactericidal mechanism.

**Figure 1.** Antimicrobial activity [bars] against *L. monocytogenes* ATCC19115 of *L. casei* AP8 [A] and *L. plantarum* H5 [B] observed during growth in MRS medium [●] and expressed in AU/ml. Results are represent the mean of three independent experiments.

To investigate the reduction of viable cells of target organism in presence of inhibitory substances, twenty mL of each filter-sterilized bacteriocin-containing cell-free supernatant were added to 100 mL of *L. monocytogenes* ATCC 19115 (4 h old at 30°C). The optical density at 600 nm and viable cell count were determined every hour during 24 h. In the control samples inoculated with indicator strain the viable cell count reached to 1011CFU/ml after 24 h incubation at 37°C. The inhibition kinetics using the bacteriocin AP8 (Figure 2) indicated a bactericidal mode of action against *L. monocytogenes*. Addition of the bacteriocin *L. casei* AP8 to early logarithmic-phase cells of indicator strain resulted in grows inhibition after 1h, followed by complete growth inhibition (slow decline) for the remaining time (20 h). In the case of *L. plantarum* H5 bacteriocin the inhibition kinetics showed a bacteriostatic mode of inhibition against indicator strain. Addition of bacteriocin H5 to culture of *L. monocytogenes* showed a growth inhibition after 1 h followed by slow growth. Experiment with stationaryphase cells did not showed any inhibition. No increase in the activity of bacteriocin AP8 and H5 were observed after treatment of the producer cells with 100 mmol/l NaCl at low pH, suggesting that these bacteriocins do not adhere to the surfaces of the producer cells.

Selection of *Lactobacillus* Species from Intestinal Microbiota of Fish for Their Potential Use as Biopreservatives 417

**Figure 3.** Tricine-SDS-PAGE gel of partially purified bacteriocins [precipitated by 60% saturated ammonium sulphate] *L. casei* AP8 [A] and *L. plantarum* H5 [B] along with the standard MW markers. The gel was overlaid with *L. monocytogenes* ATCC 19115 [approx. 106 CFU/ml], embedded in BHI agar,

B A

constitutes above 40% of the weight of the mucus [42] or higher values [41].

It could be shown that two strains, *Lactobacillus casei* AP8 and *Lactobacillus plantarum* H5 isolated from intestinal bacterial flora of beluga (*Huso huso*) and Persian sturgeon (*Acipenser persicus*) were able to produce antibacterial substances. According to the findings it was likely that the antibacterial effect was due to the formation of bacteriocin. Results from enzyme inactivation studies demonstrated that antimicrobial activity of isolates AP 8 and H5 was lost or unstable after treatment with all the proteolytic enzymes, confirming the protein status of metabolites and indicating the presence of bacteriocins. Furthermore,

Iran. Presumptive lactobacilli species found in this study were relatively similar to the species described by Bucio Galindo et al. [28]. These authors reported *L. alimentarius, L. coryneformis, L. casei, L. sakei, L. pentosus, L. plantarum, L. brevis and L. oris,* as lactobacilli presented in the intestinal content of studied fish. However, the fish species analysed in that study were different from the two species in this study which were collected from a lake environment. The biochemical characteristics used for identification of *Lactobacillus* may suggest some ideas in relation to the occurrence of the strains in nature. Most of *Lactobacillus* examined in this study (80%) had the capacity to ferment lactose and galactose. Generally, most lactobacilli are able to ferment lactose, by uptake of this disaccharide by a specific permease and splitting it by S-galactosidase for further phosphorylation of galactose and glucose [12]. Because, lactose is only present in milk and milk derivates, it is possible that these strains have evolved from environments related with mammals, as was suggested for other lactose positive *Lactobacillus* [40]. Lactose may be present or was present in the environment as a waste; resulting from livestock production, and disposal effluents from dairy factories. Another component, often fermented by the strains was the amino-sugar Nacetyl-glucosamine, a compound present in peptidoglycans, in blood, chitin and as one of the main constituents of mucus in the gastrointestinal tract [41]. The carbohydrate portion

after incubation at 30 °C for 24.

**Figure 2.** Antimicrobial effect of the CFS of *L. casei* AP8 [▲] and *L. plantarum* H5 [●] on the growth of *L. monocytogenes* ATCC 19115 at 30°C. Growth of *L. monocytogenes* ATCC 19115 without added bacteriocins [control, ♦].

### **3.6. Partial purication and molecular size of bacteriocins AP8 and H5**

Ammonium sulfate precipitation method with 60% saturated ammonium sulphate is used for partial purification of both bacteriocins. Results showed an increase (10-15%) in the inhibitory activity of both bacteriocins against *L. monocytogenes* ATCC 19115 after precipitation. The SDS-PAGE analysis of the partially purified samples showed peptide bands for bacteriocins AP8 and H5 in size of approximately 5 and 3 kDa respectively (Figure 3).

### **4. Discussion**

In this study, we isolated, quantified and characterized *Lactobacillus* species from two species of sturgeon fish inhabiting Caspian Sea to make a bank collection of strain for further research. These fishes are highly valuable species for fisheries and aquaculture in

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

[control, ♦].

**4. Discussion** 

To investigate the reduction of viable cells of target organism in presence of inhibitory substances, twenty mL of each filter-sterilized bacteriocin-containing cell-free supernatant were added to 100 mL of *L. monocytogenes* ATCC 19115 (4 h old at 30°C). The optical density at 600 nm and viable cell count were determined every hour during 24 h. In the control samples inoculated with indicator strain the viable cell count reached to 1011CFU/ml after 24 h incubation at 37°C. The inhibition kinetics using the bacteriocin AP8 (Figure 2) indicated a bactericidal mode of action against *L. monocytogenes*. Addition of the bacteriocin *L. casei* AP8 to early logarithmic-phase cells of indicator strain resulted in grows inhibition after 1h, followed by complete growth inhibition (slow decline) for the remaining time (20 h). In the case of *L. plantarum* H5 bacteriocin the inhibition kinetics showed a bacteriostatic mode of inhibition against indicator strain. Addition of bacteriocin H5 to culture of *L. monocytogenes* showed a growth inhibition after 1 h followed by slow growth. Experiment with stationaryphase cells did not showed any inhibition. No increase in the activity of bacteriocin AP8 and H5 were observed after treatment of the producer cells with 100 mmol/l NaCl at low pH,

suggesting that these bacteriocins do not adhere to the surfaces of the producer cells.

**Figure 2.** Antimicrobial effect of the CFS of *L. casei* AP8 [▲] and *L. plantarum* H5 [●] on the growth of *L. monocytogenes* ATCC 19115 at 30°C. Growth of *L. monocytogenes* ATCC 19115 without added bacteriocins

Ammonium sulfate precipitation method with 60% saturated ammonium sulphate is used for partial purification of both bacteriocins. Results showed an increase (10-15%) in the inhibitory activity of both bacteriocins against *L. monocytogenes* ATCC 19115 after precipitation. The SDS-PAGE analysis of the partially purified samples showed peptide bands for bacteriocins AP8

In this study, we isolated, quantified and characterized *Lactobacillus* species from two species of sturgeon fish inhabiting Caspian Sea to make a bank collection of strain for further research. These fishes are highly valuable species for fisheries and aquaculture in

**3.6. Partial purication and molecular size of bacteriocins AP8 and H5** 

and H5 in size of approximately 5 and 3 kDa respectively (Figure 3).

**Figure 3.** Tricine-SDS-PAGE gel of partially purified bacteriocins [precipitated by 60% saturated ammonium sulphate] *L. casei* AP8 [A] and *L. plantarum* H5 [B] along with the standard MW markers. The gel was overlaid with *L. monocytogenes* ATCC 19115 [approx. 106 CFU/ml], embedded in BHI agar, after incubation at 30 °C for 24.

Iran. Presumptive lactobacilli species found in this study were relatively similar to the species described by Bucio Galindo et al. [28]. These authors reported *L. alimentarius, L. coryneformis, L. casei, L. sakei, L. pentosus, L. plantarum, L. brevis and L. oris,* as lactobacilli presented in the intestinal content of studied fish. However, the fish species analysed in that study were different from the two species in this study which were collected from a lake environment. The biochemical characteristics used for identification of *Lactobacillus* may suggest some ideas in relation to the occurrence of the strains in nature. Most of *Lactobacillus* examined in this study (80%) had the capacity to ferment lactose and galactose. Generally, most lactobacilli are able to ferment lactose, by uptake of this disaccharide by a specific permease and splitting it by S-galactosidase for further phosphorylation of galactose and glucose [12]. Because, lactose is only present in milk and milk derivates, it is possible that these strains have evolved from environments related with mammals, as was suggested for other lactose positive *Lactobacillus* [40]. Lactose may be present or was present in the environment as a waste; resulting from livestock production, and disposal effluents from dairy factories. Another component, often fermented by the strains was the amino-sugar Nacetyl-glucosamine, a compound present in peptidoglycans, in blood, chitin and as one of the main constituents of mucus in the gastrointestinal tract [41]. The carbohydrate portion constitutes above 40% of the weight of the mucus [42] or higher values [41].

It could be shown that two strains, *Lactobacillus casei* AP8 and *Lactobacillus plantarum* H5 isolated from intestinal bacterial flora of beluga (*Huso huso*) and Persian sturgeon (*Acipenser persicus*) were able to produce antibacterial substances. According to the findings it was likely that the antibacterial effect was due to the formation of bacteriocin. Results from enzyme inactivation studies demonstrated that antimicrobial activity of isolates AP 8 and H5 was lost or unstable after treatment with all the proteolytic enzymes, confirming the protein status of metabolites and indicating the presence of bacteriocins. Furthermore, treatment with lipolytic or glycolytic enzymes did not affect the activity of antimicrobial compound produced by strain, suggesting that produced bacteriocins do not belong to the controversial group IV of the bacteriocins, which contain carbohydrates or lipids in the active molecule structure [45-47]. It is important to note that, their activities were not due to hydrogen peroxide or acidity, as antimicrobial activity was not lost after treatment with catalase. Both of the presumptive were considered to be heat stable. Although heat stability of antibacterial substances produced by *Lactobacillus* spp. has been well established [39,48,49,50-53] heat stability of *L. casei* AP8 121ºC for 10 min is novel. The result of pH stability were not coherent with previous report that had indicated the tolerance of bacteriocins to acidic pH rather than alkaline [36,54]. The loss of antimicrobial activity of AP8 bacteriocin at pH > 10 might be ascribed to proteolytic degradation, protein aggregation or instability of proteins at this extreme pH [39,48,55]. *L. casei* AP8 bacteriocin showed an increase in the inhibitory activity after treatments with SDS and EDTA, may be due to the ability of these compounds to break down the proteinaceous complex from its large form into smaller more active unite [21]. Similar to Lactocin RN78 and Plantaricin LC74, both bacteriocin *L. casei* AP8 and *L. plantarum* H5 were found to be stable after treatment with organic solvents like butanol, ethanol and methanol confirming their proteinaceous and soluble nature [18,21,56]. Pronounced inhibitory potential against various species of Grampositive bacteria were shown, including pathogenic and spoilage microorganisms such as *A. hydrophila, A. salmonicida, C. perfringens*, *B. cereus* and *L. monocytogenes*. Observed effects were consistent with reports about bacteriocins produced by other strains of LAB [1,3,17,19,20,25,41,49,55,57 59,60]. Although bacteriocins from LAB usually are ineffective against Gram-negative bacteria and rather relate to a narrow antimicrobial spectrum [9,51,53], both presumptive bacteriocins AP8 and H5 showed broad antimicrobial activity against several genera of Gram-positive and Gram-negative bacteria. Even representatives of *Pseudomonas, Salmonella, E. coli, A. hydrophila*, *A. salmonicida and V. anguillarum* could be inhibited. Moreover a high level of inhibitory activity against *Listeria monocytogenes* was observed. Earlier studied have shown that several marine bacteria may produce inhibitory substances against bacterial pathogens in aquaculture systems [1,16,19]. Hence the use of such bacteria releasing antimicrobial substances in now gaining importance in fish farming as a natural alternative to administration of antibiotics [1,61-63]. In kinetic studies, both crude bacteriocins were continuously produced during logarithmic phase followed by optimal production during stationary growth phase, suggesting that these peptides may be secondary metabolites. Similar results were reported for some bacteriocins produced by some LAB isolates [5,64,65] and is contrary for other *Lactobacillus* species bacteriocins [1,16,25,55,60,66]. Bacteriocin H5 showed a decrease in activity towards the end of stationary growth may be due to proteolytic degration, protein aggregation, and feedback regulation as has been observed for Lactacin ST13BR, Lactacin B, Helveticin J and Enterocin1146 [53,55,67]. *L. casei* AP8 crude bacteriocin demonstrated a bactericidal mode of action, as the immediate decrease in the optical density of *L. monocytogenes* was observed in mix culture. In the case of H5 bacteriocin a bacteriostatic mode of action was observed. Crud H5 bacteriocin showed a growth inhibition, followed by decrease activity for remained time, Selection of *Lactobacillus* Species from Intestinal Microbiota of Fish for Their Potential Use as Biopreservatives 419

suggesting that indicator organism became resistant to the bacteriocin or bacteriocin was destroyed by proteolytic enzymes [55]. Treating of bacteriocins AP 8 and H5 with NaCl at low pH did not result in increased levels of antilisterial activity, suggesting no adsorption of bacteriocins to their producer cells in agreement with result reported before for *Lactobacillus*

More accurate techniques could be used to determine the molecular mass of molecules, yet the SDS-PAGE technique provides valuable information about the presence of the peptides [3]. In recent years, a large number of new bacteriocins produced by *L. plantarum* have been identied and characterized and the molecular masses of all the bacteriocins produced have been reported in the range of 3-10 kDa [5,39,55]. However, to our knowledge, there is no bacteriocin produced by any *L. casei* strain with a molecular mass of 5 kDa with similar characteristics to strain investigated in this study. Thus, it is possible that this bacteriocin may be a novel bacteriocin produced by *L. casei*. The physiochemical properties of bacteriocins from *L. casei* AP8 and *L. plantarum* H5 were similar to those of other bacteriocins of lactobacilli belonging to the group IIa lactic acid bacteria with respect to molecular weight, heat and pH stability and also sensivity to proteolytic enzymes [9,45,51]. Characteristics unifying all members of class IIa bacteriocins are 1) below 10kDa [1] their potent activity against *Listeria* spp., 2) their resistance to elevated temperatures and extreme pHs, and 3) their cystibiotic feature attributed to the presence of at least one disulfide bridge, which is crucial for antibacterial activity [15,45,51,55]. Class IIa bacteriocins were formerly considered as "narrow"-spectrum antibiotics, with antimicrobial activity directed against related strains. Recently, some class IIa bacteriocins, such as bacteriocin OR-7, enterocin E50-52, and enterocin E760, have been shown to be active against both Gramnegative and Gram-positive bacteria, including *Campylobacter jejuni*, *Yersinia* spp., *Salmonella*  spp., *Escherichia coli* O157:H7, *Shigella dysenteriae*, *Staphylococcus aureus*, and *Listeria* spp. [15,

Bacteriocins AP8 and H5 showed a wide spectrum of antibacterial activity against seafood borne pathogens like *Listeria*, *Clostridium*, *Bacillus* spp, *S. aureus* and even Gram-negative pathogens like *Pseudomonas, Salmonella* and *E. coli*. Some of these foodborne pathogens can produce toxins resulting in human illness. In addition to the broad inhibition spectrum, theirs technological properties and especially cold, heat and storage stability, indicate that bacteriocins AP8 and H5 have potential for application not only as biopreservative agents to control pathogens in food products that are pasteurized and cook-chilled but also as bioprotect compounds at aquaculture. Accordingly *L. casei* may be of great interest as probiotics strains because of their ability to adhere to intestinal epithelial cells and being of human origin. Several authors have reported the production of bacteriocins by *L. casei* and *L. plantarum* strains from plant, dairy or meat origin. However, very few bacteriocins from *L. plantarum* have been reported to be isolated from fish and also based on our knowledge this

is the first report of a *L. casei* bacteriocin isolated from fish.

strains bacteriocins [55,64,66].

54, 45, 51, 3].

**5. Conclusion** 

suggesting that indicator organism became resistant to the bacteriocin or bacteriocin was destroyed by proteolytic enzymes [55]. Treating of bacteriocins AP 8 and H5 with NaCl at low pH did not result in increased levels of antilisterial activity, suggesting no adsorption of bacteriocins to their producer cells in agreement with result reported before for *Lactobacillus* strains bacteriocins [55,64,66].

More accurate techniques could be used to determine the molecular mass of molecules, yet the SDS-PAGE technique provides valuable information about the presence of the peptides [3]. In recent years, a large number of new bacteriocins produced by *L. plantarum* have been identied and characterized and the molecular masses of all the bacteriocins produced have been reported in the range of 3-10 kDa [5,39,55]. However, to our knowledge, there is no bacteriocin produced by any *L. casei* strain with a molecular mass of 5 kDa with similar characteristics to strain investigated in this study. Thus, it is possible that this bacteriocin may be a novel bacteriocin produced by *L. casei*. The physiochemical properties of bacteriocins from *L. casei* AP8 and *L. plantarum* H5 were similar to those of other bacteriocins of lactobacilli belonging to the group IIa lactic acid bacteria with respect to molecular weight, heat and pH stability and also sensivity to proteolytic enzymes [9,45,51]. Characteristics unifying all members of class IIa bacteriocins are 1) below 10kDa [1] their potent activity against *Listeria* spp., 2) their resistance to elevated temperatures and extreme pHs, and 3) their cystibiotic feature attributed to the presence of at least one disulfide bridge, which is crucial for antibacterial activity [15,45,51,55]. Class IIa bacteriocins were formerly considered as "narrow"-spectrum antibiotics, with antimicrobial activity directed against related strains. Recently, some class IIa bacteriocins, such as bacteriocin OR-7, enterocin E50-52, and enterocin E760, have been shown to be active against both Gramnegative and Gram-positive bacteria, including *Campylobacter jejuni*, *Yersinia* spp., *Salmonella*  spp., *Escherichia coli* O157:H7, *Shigella dysenteriae*, *Staphylococcus aureus*, and *Listeria* spp. [15, 54, 45, 51, 3].
