**1. Introduction**

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

Prot. 72: 365-374.

[144] Tomé E, Pereira VL, Lopes CI, Gibbs PA, Teixeira PC (2008) In vitro tests of suitability of bacteriocin-producing lactic acid bacteria, as potential biopreservation cultures in

[145] Matamoros S, Leroi F, Cardinal M, Gigout F, Kasbi Chadli F, Cornet J, Prevost F, Pilet M.F (2009). Psychrotrophic lactic acid bacteria used to improve the safety and quality of vacuum-packaged cooked and peeled tropical shrimp and cold smoked salmon. J. Food

vacuum-packaged cold-smoked salmon. Food Control. 19: 535-543.

Despite recent advances in seafood production, seafood safety is still an important public health issue. It is clear that indigenous bacteria present in marine environment as well as resulting from post contamination during processing are responsible for many cases of illnesses [1-3]. In the last years, traditional processes applied to seafood like salting, smoking and canning have decreased in favor of mild technologies involving lower salt content, lower heating temperature and vacuum (VP) or modified atmosphere packing (MAP, 3-5]. Most of these treatments are usually not sufficient to destroy microorganisms and in some cases psychrotolerant pathogenic such as *Listeria monocytogenes* or spoilage causing bacteria can develop during prolonged shelf-life of these products [2,5,6]. As several of these products are eaten raw, it is therefore essential that adequate precautious and preservation technologies are applied to maintain their safety and quality. Among alternative preservation technologies, particular attention has been paid to biopreservation to extend the shelf-life and to enhance the hygienic quality of perishable food products such as seafood, thereby minimizing the impact on nutritional and organoleptic properties [1,7,8]. In this context, lactic acid bacteria (LAB) possess a major potential in biopreservation strategies, since they are safe to consume, and during storage they naturally dominate the microbiota of many foods [7-11]. Lactic acid bacteria are gram-positive, non-sporulating and catalase negative rods or cocci that ferment various carbohydrates mainly to lactate and acetate [12]. Accordingly, they are commonly associated with nutritious environments like foods, decaying material and the mucosal surfaces of the gastrointestinal and urogenital tract [12- 14], where they enhance the host protection against pathogens [13]. Their antagonistic and inhibitory properties are due to the competition for nutrients and the production of one or more antimicrobially active metabolites such as organic acids (lactic

© 2013 Ghanbari et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and acetic acid), hydrogen peroxide, and antimicrobial peptides like bacteriocins [8-11,15- 17]. Bacteriocins are ribosomally synthesized peptides that exert their antimicrobial activity against either strains of the same species as the bacteriocin producer (narrow range), or to more distantly related species (broad range) [7,15,18]. An important reason for research on LAB based bacteriocins is due to their activity at nanomolar concentrations against number of bacterial pathogens [1,3,5,6,19,20]. Some bacteriocins even exhibit their activities against multidrug-resistant nosocomial pathogens such as methicillin-resistant *Staphylococcus aureus* (MRSA) and vancomycin-resistant enterococci [VRE, 17, 21]. Thus they also may have some big potential in medical and veterinary applications. Fermented food and plant material have been a well-known source for bacteriocin-producing LAB, but isolates from the intestinal of animals and humans has become an increasingly important source for such strains due to an increased awareness of their importance as probiotics. In fish the presence of LAB is meanwhile well documented and the bio-protective potential of some strains and/or their bacteriocin has been highlighted in the last years [4-6,16,18,22-26]. Kvasnikov et al. [12] described the presence of lactic acid bacteria, including *Lactobacillus* in the intestines of various fish species at larval, fry and fingerling stages inhabiting ponds in Ukraine. They give information on the changes in their composition as a function of the season of the year and life-stage of the fish. However, it was discussed that some human activities like artificial feeding in ponds would have had an effect on the bacterial composition and load in some fish, like carp (*Cyprinus carpio*) which showed the highest content of lactic acid bacteria in the intestines. Cai et al. [27] described the lactic acid bacteria in *Cyprinus carpio* collected from the Thajin river in Thailand. They reported the presence of *Enterococcus* spp. and the dominance of *Lactococcus garviae*, an emerging zoonotic pathogen, in *Cyprinus carpio*. Bucio Galindo et al. [23] studied the distribution of lactobacilli in the intestinal content of river fish and reported that various species of lactobacilli were present in relatively high numbers in the intestines of edible freshwater fish from the river, especially in warm season but in low numbers in cold season. There are no reports on the presence of *Lactobacillus* in the intestines of sturgeon fish inhabiting Caspian Sea, whereas other groups of bacteria have been studied in more details. In comparison with other food products of dairy or meat origin, only few bacteriocinogenic LAB strains have been recovered from seafood. The present study focuses on the characterization of antimicrobial compounds produced by the lactobacilli isolates, in addition, their ability to inhibit the growth of relevant food borne pathogens as well as of spoilage bacteria and last but not least, of contaminants in aquaculture.

Selection of *Lactobacillus* Species from Intestinal Microbiota of Fish for Their Potential Use as Biopreservatives 407

was opened with sterile scissors. After dissecting the fish, 1 g of the intestinal tract content of each fish was removed under aseptic condition and placed into previously weighed flasks

Intestinal content was homogenized in a storage medium using a vortex mixer. One milliliter was transferred to reduced neutralized bacterial peptone (NBP, Oxoid L34, Hampshire, England) 0.5 g/L, NaCl 8 g/L, cysteine.HCl 0.5 g/L, pH adjusted to 6.7 [29]. Afterwards serial dilutions were spread on plates of selective media and incubated at the following conditions. Columbia blood agar (CAB, Oxoid CM 331) was used as a selective medium to make an estimation of the cultivable total anaerobic counts [29]. All the inoculated plates were incubated anaerobically at 30°C for 48 h. The following two media were used to isolate lactic acid bacteria (LAB). MRS (MRS, Merck, Darmstadt, Germany) with 1.5% agar (M641, HiMedia, Mumbai, India) and pH adjusted to 4.2 (MRS 4.2) and incubated anaerobically at 30°C for 96 h was used as a selective medium for lactic acid bacteria. MRS is an inhibitory medium for *Carnobacterium*. Anaerobic MRS with Vancomycin and Bromocresol green (LAMVAB), incubated at 30°C for 96 h was used as an elective and selective medium for *Lactobacillus* spp. [30]. Anaerobic incubation of the three media was made in an anaerobic Gas-Pack system (LE002, HiMedia, Mumbai, India) with a mixture of 80% N2, 10% H2 and 10% CO2. Colonies were selected either randomly, or in case of less than 10 colonies per each plate, all the samples were counted according to the method described by Thapa et al. [31]. Purity of the isolates was checked again by streaking them onto fresh agar plates of the isolation media, followed by microscopic examinations. Identified strains

of lactobacilli were kept in MRS broth with 15% (v/v) glycerol at -20°C.

Eighty four strains were randomly selected for identification procedures based on the phenotypical characteristics. Cell morphology and motility of all isolates were observed using a phase contrast microscope (CH3-BH-PC, Olympus, Japan). Isolates were gramstained and tested for catalase production test. Preliminary identification and grouping was based on the cell morphology and phenotypic properties such as CO2 production from glucose, hydrolysis of arginine, growth at different temperatures (10, 15 and 45°C), and at different pH (3.9 and 9.6). As well as the ability to grow in different concentrations of NaCl (6.5% (w/v), 10% (w/v) and 18% (w/v)) in MRS broth was checked as well. The configuration of lactic acid produced from glucose was determined enzymatically using d-lactate and llactate dehydrogenase test kits (Roche Diagnostic, France). The presence of diaminopimelic acid (DAP) in the cell walls of LAB was determined using thin-chromatography on cellulose plates. Fermentation of carbohydrates was determined using API 50 CHL (API 50 CH is a standardized system, associating 50 biochemical tests for the study of carbohydrate metabolism in microorganisms. API 50 CH is used in conjunction with API 50 CHL Medium for the identification of *Lactobacillus* and related genera) strips according to the

**2.3. Characterization procedures for lactic acid bacteria** 

containing storage medium.

**2.2. Media and culture condition** 

### **2. Materials and methods**

### **2.1. Fish intestine samples**

Two species of Persian sturgeon (*Acipenser persicus*) and Beluga (*Huso huso*) were collected from the south coast of Caspian Sea in Iran. Twenty two individuals of these fish in adult stage were selected. The weight and length of the fish were measured before dissection. The fish were sacrificed by physical destruction of the brain, and the number of incidental organisms was reduced by washing the fish skin with 70% ethanol. Then, the ventral surface was opened with sterile scissors. After dissecting the fish, 1 g of the intestinal tract content of each fish was removed under aseptic condition and placed into previously weighed flasks containing storage medium.

### **2.2. Media and culture condition**

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

and acetic acid), hydrogen peroxide, and antimicrobial peptides like bacteriocins [8-11,15- 17]. Bacteriocins are ribosomally synthesized peptides that exert their antimicrobial activity against either strains of the same species as the bacteriocin producer (narrow range), or to more distantly related species (broad range) [7,15,18]. An important reason for research on LAB based bacteriocins is due to their activity at nanomolar concentrations against number of bacterial pathogens [1,3,5,6,19,20]. Some bacteriocins even exhibit their activities against multidrug-resistant nosocomial pathogens such as methicillin-resistant *Staphylococcus aureus* (MRSA) and vancomycin-resistant enterococci [VRE, 17, 21]. Thus they also may have some big potential in medical and veterinary applications. Fermented food and plant material have been a well-known source for bacteriocin-producing LAB, but isolates from the intestinal of animals and humans has become an increasingly important source for such strains due to an increased awareness of their importance as probiotics. In fish the presence of LAB is meanwhile well documented and the bio-protective potential of some strains and/or their bacteriocin has been highlighted in the last years [4-6,16,18,22-26]. Kvasnikov et al. [12] described the presence of lactic acid bacteria, including *Lactobacillus* in the intestines of various fish species at larval, fry and fingerling stages inhabiting ponds in Ukraine. They give information on the changes in their composition as a function of the season of the year and life-stage of the fish. However, it was discussed that some human activities like artificial feeding in ponds would have had an effect on the bacterial composition and load in some fish, like carp (*Cyprinus carpio*) which showed the highest content of lactic acid bacteria in the intestines. Cai et al. [27] described the lactic acid bacteria in *Cyprinus carpio* collected from the Thajin river in Thailand. They reported the presence of *Enterococcus* spp. and the dominance of *Lactococcus garviae*, an emerging zoonotic pathogen, in *Cyprinus carpio*. Bucio Galindo et al. [23] studied the distribution of lactobacilli in the intestinal content of river fish and reported that various species of lactobacilli were present in relatively high numbers in the intestines of edible freshwater fish from the river, especially in warm season but in low numbers in cold season. There are no reports on the presence of *Lactobacillus* in the intestines of sturgeon fish inhabiting Caspian Sea, whereas other groups of bacteria have been studied in more details. In comparison with other food products of dairy or meat origin, only few bacteriocinogenic LAB strains have been recovered from seafood. The present study focuses on the characterization of antimicrobial compounds produced by the lactobacilli isolates, in addition, their ability to inhibit the growth of relevant food borne pathogens as well as of

spoilage bacteria and last but not least, of contaminants in aquaculture.

Two species of Persian sturgeon (*Acipenser persicus*) and Beluga (*Huso huso*) were collected from the south coast of Caspian Sea in Iran. Twenty two individuals of these fish in adult stage were selected. The weight and length of the fish were measured before dissection. The fish were sacrificed by physical destruction of the brain, and the number of incidental organisms was reduced by washing the fish skin with 70% ethanol. Then, the ventral surface

**2. Materials and methods** 

**2.1. Fish intestine samples** 

Intestinal content was homogenized in a storage medium using a vortex mixer. One milliliter was transferred to reduced neutralized bacterial peptone (NBP, Oxoid L34, Hampshire, England) 0.5 g/L, NaCl 8 g/L, cysteine.HCl 0.5 g/L, pH adjusted to 6.7 [29]. Afterwards serial dilutions were spread on plates of selective media and incubated at the following conditions. Columbia blood agar (CAB, Oxoid CM 331) was used as a selective medium to make an estimation of the cultivable total anaerobic counts [29]. All the inoculated plates were incubated anaerobically at 30°C for 48 h. The following two media were used to isolate lactic acid bacteria (LAB). MRS (MRS, Merck, Darmstadt, Germany) with 1.5% agar (M641, HiMedia, Mumbai, India) and pH adjusted to 4.2 (MRS 4.2) and incubated anaerobically at 30°C for 96 h was used as a selective medium for lactic acid bacteria. MRS is an inhibitory medium for *Carnobacterium*. Anaerobic MRS with Vancomycin and Bromocresol green (LAMVAB), incubated at 30°C for 96 h was used as an elective and selective medium for *Lactobacillus* spp. [30]. Anaerobic incubation of the three media was made in an anaerobic Gas-Pack system (LE002, HiMedia, Mumbai, India) with a mixture of 80% N2, 10% H2 and 10% CO2. Colonies were selected either randomly, or in case of less than 10 colonies per each plate, all the samples were counted according to the method described by Thapa et al. [31]. Purity of the isolates was checked again by streaking them onto fresh agar plates of the isolation media, followed by microscopic examinations. Identified strains of lactobacilli were kept in MRS broth with 15% (v/v) glycerol at -20°C.

### **2.3. Characterization procedures for lactic acid bacteria**

Eighty four strains were randomly selected for identification procedures based on the phenotypical characteristics. Cell morphology and motility of all isolates were observed using a phase contrast microscope (CH3-BH-PC, Olympus, Japan). Isolates were gramstained and tested for catalase production test. Preliminary identification and grouping was based on the cell morphology and phenotypic properties such as CO2 production from glucose, hydrolysis of arginine, growth at different temperatures (10, 15 and 45°C), and at different pH (3.9 and 9.6). As well as the ability to grow in different concentrations of NaCl (6.5% (w/v), 10% (w/v) and 18% (w/v)) in MRS broth was checked as well. The configuration of lactic acid produced from glucose was determined enzymatically using d-lactate and llactate dehydrogenase test kits (Roche Diagnostic, France). The presence of diaminopimelic acid (DAP) in the cell walls of LAB was determined using thin-chromatography on cellulose plates. Fermentation of carbohydrates was determined using API 50 CHL (API 50 CH is a standardized system, associating 50 biochemical tests for the study of carbohydrate metabolism in microorganisms. API 50 CH is used in conjunction with API 50 CHL Medium for the identification of *Lactobacillus* and related genera) strips according to the 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] and Hammes and Vogel [32].

Selection of *Lactobacillus* Species from Intestinal Microbiota of Fish for Their Potential Use as Biopreservatives 409

activity was determined by adjusting the pH of the CFS (cell free supernatant (pH 6.5) of 24-h lactobacilli cultures incubated at 30°C) with diluted appropriate volumes of HCl and NaOH (Table 3). After incubating for 2 h, the pH of the samples was readjusted to 6.5 followed by sterilization (0.2 µm, Sigma, UK). In all cases, the remaining bacteriocin activity was assessed exemplarily by using strain *L. monocytogenes* ATCC 19115 as the indicator bacterium and by applying the agar disk diffusion plate bioassay. Untreated cell-free supernatants were used as

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.

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

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

with sterile 1 M NaOH followed by testing the bacteriocin activity as described above.

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

controls and experiments were performed in duplicate.

Indicator cells without CFSs were used as control.

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

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