**5. LAB in fish and fish products**

LAB are not considered as genuine microflora of the aquatic environment, but certain genera, including *Carnobacterium, Lactobacillus, Enterococcus,* and *Lactococcus,* have been found in fresh and sea water fresh fish [61,63,79-83]. The number of lactobacilli in the gastrointestinal tract of Arctic char was smaller in those reared in sea water than in fresh water, while the number of *Leuconostoc* and enterococci remained the same [84]. It is well documented that lactobacilli are part, not dominant, of the native intestinal microbiota of Arctic charr (*Salvelinus alpinus* L.), Atlantic cod, Atlantic salmon (*Salmo salar* L.), and brown trout (*Salmo trutta*) [82,85]. Several studies have shown the presence of other lactic acid bacteria, specially carnobacteria such as *Carnobacterium maltaromaticum* and *Carnobacterium divergence* within the intestinal content of salmonid species like Arctic charr (*Salvelinus alpinus*), Atlantic salmon (*Salmo salar*), rainbow trout (*Oncorhyncus mykiss*) [63,86-89], Atlantic cod [89], common wolffish (*Anarhichas lupus* L.) [85], brown trout [82] and also wild pike [63,82]. Bacteria of the genus *Enterococcus* have been isolated from the intestine of common carp (*Cyprinus carpio*) and brown trout [80,82].

LAB dominating in spoiled vacuum-packaged cold-smoked fish products include the genera of *Lactobacillus*, *Leuconostoc*, *Lactococcus* and *Carnobacterium* [9]. Magnússon & Traustadóttir [91] reported the complete dominance of homofermentative lactobacilli in vacuumpackaged cold-smoked herring. In vacuum packaged cold-smoked salmon and herring, *Lactobacillus curvatus* has been found in majority together with lower numbers of *Lactobacillus sakei*, *Lactobacillus plantarum*, *Lactococcus* spp. and *Leuconostoc mesenteroides* [58,]. Paludan-Müller, Huss, & Gram [92] identified *Carnobacterium piscicola* as the dominant microorganism isolated from spoiled vacuum-packaged cold-smoked salmon. Leroi et al. [93] also isolated carnobacteria during the first stage of storage of vacuum-packaged coldsmoked salmon, whereas *Lactobacillus farciminis*, *Lactobacillus sakei*, and *Lactobacillus alimentarius* were isolated at advanced storage times. Other studies have also confirmed that most bacteria in vacuum-packaged "gravad" fish products stored at refrigeration temperatures are carnobacteria [94] and *L. sakei*, and to a lesser extent *Leuconostoc* spp., *L. curvatus,* and *Weissella viridescens* [95]. Gancel et al [90] have isolated 78 strains belonging to the genus *Lactobacillus* from fillets of vacuum packed smoked and salted herring *(Clupea harengus).* LAB has been found to occur in marinated herring, herring fillets and cured stockfish [58]. In marinated or dried fish, the lactic acid bacteria flora maybe quite diverse since the presence of *Lactobacilli* and *Pediococci* has been reported [90]. Thai fermented fishery products were screened for the presence of LAB by Ostergaard et al. [96]. LAB was found to occur in the low salted fermented products in the range of 107-109 cfu/g. The high salt product "hoi dorng" had a lower LAB count of 103-105 cfu/g. Olympia et al [97] have isolated 108 LAB/g from a Philippine low salt rice-fish product burong bangus. Several studies have been mentioned that some species of *Carnobacteriuim* such as *C. divergens* and *C. maltaromaticum* are present in seafood and are able to grow to high concentrations in different fresh and lightly preserved products such as modified atmosphere-packed (MAP) [98-100], chilled MAP [101,102], high-pressure processing treated seafood products [103] and vacuum-packed cold smoked or sugar-salted ('gravad') seafood [53,93,95]. These studies clearly highlight the ability of LAB fish isolates to grow on different harsh condition rather than other organisms. Obviously many investigation have been shown that carnobacteria are common in chilled fresh and lightly preserved seafood, but at higher storage temperatures (15–25°C) other species could be dominate the spoilage microbial community of seafood.

### **6. Application of LAB in seafood**

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

inhibiting growth at around pH 5.1.

*4.2.3. Other antimicrobials of LAB* 

**5. LAB in fish and fish products** 

common carp (*Cyprinus carpio*) and brown trout [80,82].

slowing down metabolic activities, and in the case of Enterobacteriaceae such as *E. coli* 

Hydrogen peroxide is produced from lactate by LAB in the presence of oxygen as a result of the action of flavoprotein oxidases or nicotinamide adenine dinucleotide (NADH) peroxidise [76]. The antimicrobial effect of H2O2 may result from the oxidation of sulfhydryl groups causing denaturing of a number of enzymes, and from the peroxidation of membrane lipids thus increasing membrane permeability [8]. Most undesirable bacteria such as *Pseudomonas* spp. and *S. aureus* are many times sensitive to H202. Carbon dioxide (CO2) is mainly produced by heterofermentative LAB. CO2 plays a role in creating an anaerobic environment which inhibits enzymatic decarboxylations, and the accumulation of CO2 in the membrane lipid bilayer may cause a dysfunction in permeability [8]. CO2 can effectively inhibit the growth of many food spoilage microorganisms, especially Gramnegative psychrotrophic bacteria [77]. Diacetyl, an aroma component, is produced by strains within all genera of LAB by citrate fermentation. It is produced by heterofermentative lactic acid bacteria as a by-product along with lactate as the main product [8]. Diacetyl is a high value product and is extensively used in the dairy industry as a preferred flavour compound. Diacetyl also has antimicrobial properties. Diacetyl was found to be more active against gram-negative bacteria, yeasts, and molds than against gram-positive bacteria. Diacetyl is thought to react with the arginine-binding protein of gram-negative bacteria and thereby interfering with the utilization of this amino acid [78].

LAB are not considered as genuine microflora of the aquatic environment, but certain genera, including *Carnobacterium, Lactobacillus, Enterococcus,* and *Lactococcus,* have been found in fresh and sea water fresh fish [61,63,79-83]. The number of lactobacilli in the gastrointestinal tract of Arctic char was smaller in those reared in sea water than in fresh water, while the number of *Leuconostoc* and enterococci remained the same [84]. It is well documented that lactobacilli are part, not dominant, of the native intestinal microbiota of Arctic charr (*Salvelinus alpinus* L.), Atlantic cod, Atlantic salmon (*Salmo salar* L.), and brown trout (*Salmo trutta*) [82,85]. Several studies have shown the presence of other lactic acid bacteria, specially carnobacteria such as *Carnobacterium maltaromaticum* and *Carnobacterium divergence* within the intestinal content of salmonid species like Arctic charr (*Salvelinus alpinus*), Atlantic salmon (*Salmo salar*), rainbow trout (*Oncorhyncus mykiss*) [63,86-89], Atlantic cod [89], common wolffish (*Anarhichas lupus* L.) [85], brown trout [82] and also wild pike [63,82]. Bacteria of the genus *Enterococcus* have been isolated from the intestine of

LAB dominating in spoiled vacuum-packaged cold-smoked fish products include the genera of *Lactobacillus*, *Leuconostoc*, *Lactococcus* and *Carnobacterium* [9]. Magnússon & Traustadóttir Treating catfish fillets with of 0.50% sodium acetate, 0.25% potassium sorbate with 2.50% lactic acid culture completely inhibited growth of Gram negative bacteria, improved catfish odor and appearance during 13 days storage [110]. Einarsson & Lauzon [111] treated shrimps with various bacteriocins from lactic acid bacteria and reported shelf life extension except carnocin UI49. Total mesophilic and psychotropic bacteria and MRS counts of the samples treated with carnocin UI49 were not different than those of controls at 4.5°C. In a study with five strains of lactic acid bacteria (four *Lactobacillus* and one *Carnobacterium*) on fermented salmon fillets, *L. sake* LAD and *L. alimentarius* BJ33 was regarded as suitable starters for fermentation of salmon fillets [112] based on starter growth (increase of more than 1log in 3 days) and acidification of muscle (e.g. pH reduction of approximately 0.7

units in 5 days) as well as sensory evaluation. Kisla & Ünlütürk [113] studied the microbial shelf life of rainbow trout treated with nisin-containing aqueous solution of *Lactococcus lactis* subsp. lactis NCFB 497and lactic acid. They reported the dipping of rainbow trout fillets into a lactic culture did not prolonged the shelf life due to the low inoculum level and type of lactic culture used. Elotmani & Assobhei [114] evaluated the inhibition of the microbial flora of sardine by using nisin and a lactoperoxidase system (LP), observing the efficiency of the nisin–LP combination in inhibiting fish spoilage flora. In another study growth of *L. monocytogenes* was significantly inhibited (P < 0.05) by *L. sakei* Lb706 in rainbow trout fillets stored under vacuum at 4°C during 10 days of storage while bacteriocin negative Lb706-B did not affect the growth of *L. monocytogenes*. In the presence of the sakacin A-producing strain of *L*. *sakei* (Lb706), the growth of *L*. *monocytogenes* was significantly inhibited (P < 0.05) in the first 3 days of storage at 10°C, after which its count increased to 107 CFU g-1 [115]. Altieri et al. [106] succeeded in inhibiting *Pseudomonas* spp. and *P. phosphoreum* in VP fresh plaice fillets at low temperatures by using a *Bifidobacterium bifidum* starter, and extending the shelf-life, especially under MAP. Bifidobacteria combined with sodium acetate (SA) extended refrigerated shelf-life of catfish fillets at 4°C [116]. The application of two *Lactobacillus sakei* CECT 4808 and *L. curvatus* CECT 904T protective cultures on refrigerated vacuum-packed rainbow trout (*Oncorhynchus mykiss*) fillets resulted in extension of shelf-life by 5 days by significantly improved in the counts of all microbiological spoilage indicator organisms (Enterobacteriaceae, *Pseudomonas* spp., H2S-producing bacteria, yeasts and moulds) and also significantly improved in all examined chemical parameters and off-odour [117].

Lactic Acid Bacteria and Their Bacteriocins: A Promising Approach to Seafood Biopreservation 391

time of Chilled Shrimp. Fall et al. [121] evidenced the in situ inhibition of *B. thermosphacta*, a major spoiling bacterium, by *L*. *piscium* that could explain the protective effect observed in shrimp. Additionally, those strains also showed an inhibitory effect on *L. monocytogenes* [124] and *Staph. aureus*. Recently, Sudalayandi & Manja [109] succeeded to preserve fresh fish through controlling spoilage bacteria and amines of Indian mackerel fish chank for two days at 37°C by inoculating them with different strains of LAB such as *Pediococcus acidilactici*, *Pediococcus pentosaceous*, *Streptococcus thermophilus, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus acidophilus* and *Lactobacillus helveticus*. Using bacteriocin-like metabolite producer and non-producer strains of *Pediococcus* spp. [125] only slightly improved sensory quality of Horse Mackerel during cold storage. It was concluded that *Pediococcus* strains used in this study were not proper for preserving horse mackerel fillets especially at low storage temperatures. EntP-producing enterococci isolated from farmed turbot, under a spray-dried format exhibited antilisterial, antistaphylococcal, and antibacilli activities in turbot fillets either vacuum-packaged or subjected to modified-atmosphere

LAB Protective cultures have not been applied in many other seafood products except for cold smoked salmon (CSS), as they are normally flora of such products at the end of storage, and *L. monocytogenes* control. The effectiveness of bacteriocins to control growth of *L. monocytogenes* in vacuum packed cold smoked salmon has also been demonstrated by several researchers. Among them, Sakacin P has been found to be very potent against *L. monocytogenes* and is one of the most extensively studied bacteriocins [126-131]. Leroi et al. [132] succeeded in increasing the sensory use-by-date of CSS slices by inoculating them with strains of *Carnobacterium* sp. However the results varied depending on the batch treated. Addition of nisin to CO2 packed cold smoked salmon resulted in a 1 to 2 log10 reduction of *L. monocytogenes* [11]. Using a strain of *C. maltaromaticum,* Paludan-Müller et al. [92] only slightly extended the shelf-life of smoked salmon. Budu-Amoako et al. [133] tested nisin combined with heat as anti Listerial treatment in cold- packed lobster meat, finding decimal reductions of inoculated *L. monocytogenes* of 3 to 5 logs, whereas heat or nisin alone resulted

Duffes et al. [65] isolated *C. divergens* and *C. maltaromaticum* strains that exhibited listericidal activity in a model experiment with cold-smoked fish. They found that *C. piscicola* V1 inhibited *L. monocytogenes* by the in situ production of bacteriocins in vacuum-packed coldsmoked salmon stored at 4°C and 8°C. In contrast, another related species, namely, *C. divergens* V41 and its divercin V41, only exhibited a bacteriostatic effect on the target microorganism. Two strains of *C. maltaromaticum* isolated from CSS demonstrated their efficiency to limit the growth of *L. monocytogenes* in VP CSS during 31 days of storage at 5°C [134]. In a study using vacuum-packed cold smoked rainbow trout, the combination of nisin and sodium lactate injected into smoked fish decreased the count of *L. monocytogenes* from 3.3 to 1.8 log10 over 16 days of storage at 8°C [135]. Sakacin P was added to vacuum-packed cold smoked salmon, a lightly processed high-fat (15–20%) product, together with a sakacin P-producing *L. sakei* culture in order to study the effect on the growth of *L*. *monocytogenes*. In

packaging [2].

in decimal reductions of 1 to 3 logs.

Under biopreservation, combined coating of *Lactobacillus casei* DSM 120011 and *Lactobacillus acidophilus* 1M in *Streptomces* sp. NIOF metabolites, played effective role in lowering the biochemical and microbiological changes, extended shelf-life and safety of stored fish under low temperature as reported by Daboor & Ibrahim [118]. Tahiri et al. [119] suggest that selection of protective strains to improve the sensory quality of seafood products should focus on specific spoilage microorganism's inhibition. This approach was chosen by Matamoros et al., [120] who have isolated seven strains from various marine products on the basis of their activity against many spoiling and pathogenic, Gram positive and Gram negative marine bacteria. Among strains, two *Le. gelidum,* and two *Lc. piscium* demonstrated promising effect in delaying the spoilage of tropical shrimp and of VP CSS. However, no correlation with the classical quality indices measured was evidenced. A recent study demonstrated that this protective effect could be due to the inhibition of *B. thermosphacta*  identified as one of the major spoiler organisms in cooked shrimp stored under MAP [121]. The inoculation of Tilapia (*Oreochromis niloticus*) fillets with *Lactobacillus casei* DSM 120011 and *Lactobacillus acidophilus* 1M at 2% concentration decreased both total volatile basic nitrogen (TVB-N), trimethylamine nitrogen (TMA-N) and thiobarbituric acid (TBA) values and improved the biochemical quality criteria, microbial aspects and safety of frozen fish fillets during 45 and 90 days storage. [122].

For Shirazinejad et al. [123] 2.0% lactic acid combined with nisin indicated the highest reduction in population of *Pseudomonas* spp. and H2S producing bacteria during storage time of Chilled Shrimp. Fall et al. [121] evidenced the in situ inhibition of *B. thermosphacta*, a major spoiling bacterium, by *L*. *piscium* that could explain the protective effect observed in shrimp. Additionally, those strains also showed an inhibitory effect on *L. monocytogenes* [124] and *Staph. aureus*. Recently, Sudalayandi & Manja [109] succeeded to preserve fresh fish through controlling spoilage bacteria and amines of Indian mackerel fish chank for two days at 37°C by inoculating them with different strains of LAB such as *Pediococcus acidilactici*, *Pediococcus pentosaceous*, *Streptococcus thermophilus, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus acidophilus* and *Lactobacillus helveticus*. Using bacteriocin-like metabolite producer and non-producer strains of *Pediococcus* spp. [125] only slightly improved sensory quality of Horse Mackerel during cold storage. It was concluded that *Pediococcus* strains used in this study were not proper for preserving horse mackerel fillets especially at low storage temperatures. EntP-producing enterococci isolated from farmed turbot, under a spray-dried format exhibited antilisterial, antistaphylococcal, and antibacilli activities in turbot fillets either vacuum-packaged or subjected to modified-atmosphere packaging [2].

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

fillets during 45 and 90 days storage. [122].

[117].

units in 5 days) as well as sensory evaluation. Kisla & Ünlütürk [113] studied the microbial shelf life of rainbow trout treated with nisin-containing aqueous solution of *Lactococcus lactis* subsp. lactis NCFB 497and lactic acid. They reported the dipping of rainbow trout fillets into a lactic culture did not prolonged the shelf life due to the low inoculum level and type of lactic culture used. Elotmani & Assobhei [114] evaluated the inhibition of the microbial flora of sardine by using nisin and a lactoperoxidase system (LP), observing the efficiency of the nisin–LP combination in inhibiting fish spoilage flora. In another study growth of *L. monocytogenes* was significantly inhibited (P < 0.05) by *L. sakei* Lb706 in rainbow trout fillets stored under vacuum at 4°C during 10 days of storage while bacteriocin negative Lb706-B did not affect the growth of *L. monocytogenes*. In the presence of the sakacin A-producing strain of *L*. *sakei* (Lb706), the growth of *L*. *monocytogenes* was significantly inhibited (P < 0.05) in the first 3 days of storage at 10°C, after which its count increased to 107 CFU g-1 [115]. Altieri et al. [106] succeeded in inhibiting *Pseudomonas* spp. and *P. phosphoreum* in VP fresh plaice fillets at low temperatures by using a *Bifidobacterium bifidum* starter, and extending the shelf-life, especially under MAP. Bifidobacteria combined with sodium acetate (SA) extended refrigerated shelf-life of catfish fillets at 4°C [116]. The application of two *Lactobacillus sakei* CECT 4808 and *L. curvatus* CECT 904T protective cultures on refrigerated vacuum-packed rainbow trout (*Oncorhynchus mykiss*) fillets resulted in extension of shelf-life by 5 days by significantly improved in the counts of all microbiological spoilage indicator organisms (Enterobacteriaceae, *Pseudomonas* spp., H2S-producing bacteria, yeasts and moulds) and also significantly improved in all examined chemical parameters and off-odour

Under biopreservation, combined coating of *Lactobacillus casei* DSM 120011 and *Lactobacillus acidophilus* 1M in *Streptomces* sp. NIOF metabolites, played effective role in lowering the biochemical and microbiological changes, extended shelf-life and safety of stored fish under low temperature as reported by Daboor & Ibrahim [118]. Tahiri et al. [119] suggest that selection of protective strains to improve the sensory quality of seafood products should focus on specific spoilage microorganism's inhibition. This approach was chosen by Matamoros et al., [120] who have isolated seven strains from various marine products on the basis of their activity against many spoiling and pathogenic, Gram positive and Gram negative marine bacteria. Among strains, two *Le. gelidum,* and two *Lc. piscium* demonstrated promising effect in delaying the spoilage of tropical shrimp and of VP CSS. However, no correlation with the classical quality indices measured was evidenced. A recent study demonstrated that this protective effect could be due to the inhibition of *B. thermosphacta*  identified as one of the major spoiler organisms in cooked shrimp stored under MAP [121]. The inoculation of Tilapia (*Oreochromis niloticus*) fillets with *Lactobacillus casei* DSM 120011 and *Lactobacillus acidophilus* 1M at 2% concentration decreased both total volatile basic nitrogen (TVB-N), trimethylamine nitrogen (TMA-N) and thiobarbituric acid (TBA) values and improved the biochemical quality criteria, microbial aspects and safety of frozen fish

For Shirazinejad et al. [123] 2.0% lactic acid combined with nisin indicated the highest reduction in population of *Pseudomonas* spp. and H2S producing bacteria during storage LAB Protective cultures have not been applied in many other seafood products except for cold smoked salmon (CSS), as they are normally flora of such products at the end of storage, and *L. monocytogenes* control. The effectiveness of bacteriocins to control growth of *L. monocytogenes* in vacuum packed cold smoked salmon has also been demonstrated by several researchers. Among them, Sakacin P has been found to be very potent against *L. monocytogenes* and is one of the most extensively studied bacteriocins [126-131]. Leroi et al. [132] succeeded in increasing the sensory use-by-date of CSS slices by inoculating them with strains of *Carnobacterium* sp. However the results varied depending on the batch treated. Addition of nisin to CO2 packed cold smoked salmon resulted in a 1 to 2 log10 reduction of *L. monocytogenes* [11]. Using a strain of *C. maltaromaticum,* Paludan-Müller et al. [92] only slightly extended the shelf-life of smoked salmon. Budu-Amoako et al. [133] tested nisin combined with heat as anti Listerial treatment in cold- packed lobster meat, finding decimal reductions of inoculated *L. monocytogenes* of 3 to 5 logs, whereas heat or nisin alone resulted in decimal reductions of 1 to 3 logs.

Duffes et al. [65] isolated *C. divergens* and *C. maltaromaticum* strains that exhibited listericidal activity in a model experiment with cold-smoked fish. They found that *C. piscicola* V1 inhibited *L. monocytogenes* by the in situ production of bacteriocins in vacuum-packed coldsmoked salmon stored at 4°C and 8°C. In contrast, another related species, namely, *C. divergens* V41 and its divercin V41, only exhibited a bacteriostatic effect on the target microorganism. Two strains of *C. maltaromaticum* isolated from CSS demonstrated their efficiency to limit the growth of *L. monocytogenes* in VP CSS during 31 days of storage at 5°C [134]. In a study using vacuum-packed cold smoked rainbow trout, the combination of nisin and sodium lactate injected into smoked fish decreased the count of *L. monocytogenes* from 3.3 to 1.8 log10 over 16 days of storage at 8°C [135]. Sakacin P was added to vacuum-packed cold smoked salmon, a lightly processed high-fat (15–20%) product, together with a sakacin P-producing *L. sakei* culture in order to study the effect on the growth of *L*. *monocytogenes*. In

this product, the combination of puried sakacin P and a live culture was found to be bactericidal against *L. monocytogenes*. The addition of sakacin P alone inhibited the growth of *L. monocytogenes* on this product for about 1 week [126]. Silva et al. [136] used a bacteriocinproducing *Carnobacterium* strain under a spray-dried format. This strain survived the process and retained antilisterial ability, although it lost activity against other Gram-positive targets such as *Staph. aureus.* Some authors have evaluated the antimicrobial activity of nisin combined with other bacteriocins. Bouttefroy & Milliere [137] tested combinations of nisin and curvaticin 13 produced by *L. curvatus* SB13 for preventing the regrowth of bacteriocinresistant cells of *L. monocytogenes*, finding that this combination induced a greater inhibitory effect than the use of a single bacteriocin. Aasen et al. [131] studied the interactions of the bacteriocins sakacin P and nisin with food constituents in cold-smoked salmon, chicken cold cuts, and raw chicken. They stated that owing to the amphiphilic nature of these peptides, they can be adsorbed to food macromolecules and undergo proteolytic degradation, which may limit their use as preservation agents. More than 80% of the added sakacin P and nisin were rapidly adsorbed by proteins in the food matrix that had not been heat-treated, less than 1% of the total activity remaining after 1 week in cold- smoked salmon. In heat-treated foods, they found that, bacteriocin activity was stable for more than 4 weeks. No important differences were observed between sakacin P and nisin, but less nisin was adsorbed by muscle proteins at low pH. The growth of *L. monocytogenes* was completely inhibited for at least 3 weeks in both chicken cold cuts and cold-smoked salmon by the addition of sakacin P (3.5 μ/g), despite proteolytic degradation in the salmon.

Lactic Acid Bacteria and Their Bacteriocins: A Promising Approach to Seafood Biopreservation 393

effect on the inhibition of the Gram positive spoilage bacteria *B. thermosphacta* in cooked shrimps [143]. The anti-listerial activity of 3 LAB strains used individually or as co-cultures was assayed on cold-smoked salmon artificially contaminated with *L. innocua* and stored under vacuum at 4°C [142]. The association of *L. casei* T3 and *L. plantarum* PE2 was the most effective, probably due to a competition mechanism against the pathogen. In their study Tomé et al. [144] have also selected a strain of *Enterococcus faecium* among five bacteriocinogenic LAB strains for its ability to induce a decrease of the population of *L. innocua* inoculated in CSS. However in these studies the inhibition activities were not confirmed on *L. monocytogenes.* For Matamoros et al. [145] two LAB strains, *Lactococcus piscium* EU2241 and *Leuconostoc gelidum* EU2247 were efficient to limit the growth of both pathogenic bacteria *L. monocytogenes* and *S. aureus* in a challenge test in cooked shrimp stored under VP from 2 to 3 log CFU g-1 units after 4 weeks at 8°C followed by 1 week at 20°C. The strain of *Leuconostoc* produced a bacteriocin-like compound but its activity was slight lower than the *Lactococcus* strain that was non-bacteriocinogenic. In another study, the application of *C. divergens* M35 towards *L. monocytogenes* in CSS resulted in a maximal decrease of 3.1 log CFU g-1 of the pathogenic bacteria after 21 days of storage at 4°C whereas

The presence of LAB in many processed seafood product is now well documented and the bio-protective potential of many strains and/or their bacteriocin has been highlighted in the last years. In situ production is readily cost-effective provided that the bacteriocin producers are technologically suitable. To date, only nisin and pediocin PA - 1 have been applied commercially in food applications where they are used to protect against spoilage and pathogenic organisms. However, other bacteriocins could be at least as effective for food processors as it is possible to apply them with hurdle approaches, particularly in light of consumer demands for minimally processed, safe, preservative - free foods. Control of pathogenic bacteria has widely focused on *L. monocytogenes* considered as the main risk in ready-to-eat seafood. However, in these minimally processed products, the new combination of hurdles can give selective advantages to enhance food safety and quality, particularly effective against other pathogenic bacteria like clostridia, vibrio or staphylococci. These goals can be facilitated through the incorporation of live bacteriocin producing strain(s) or through the use of bacteriocins as concentrated preparations, either through direct addition to the seafood or in an immobilized form on packaging as well as in conjunction with other factors such as high pressure or pulse electric fields, to achieve more effective preservation of foods. The great results obtained with protective culture, bacteriocins for improving safety and quality of seafood products clearly indicate that the application of LAB protective culture and/or their bacteriocins in seafood product can suggest several important benefits; 1) extended shelf life of seafood during storage time, 2) decrease the risk for transmission of foodborne pathogens in lightly preserved seafood products, 3) ameliorate the economic losses due to seafood spoilage, 4) reduce the application of chemical preservatives and drastic physical treatments such as heating,

a non bacteriocinogenic strain had no effect [119].

**7. Conclusion and future prospective** 

In the presence of the bacteriocinogenic strain *C. maltaromaticum* CS526 isolated from surimi, the population of *L. monocytogenes* in CSS decreased from 103 to 50 CFU g-1 after 7 days at 4°C [138]. This activity could be linked to the production of the bacteriocin piscicocin CS526, since a non-bacteriocin producing strain had a lower effect on the growth of the pathogenic bacteria [138, 139]. The growth of the protective *Carnobacterium* strains did not modify the sensory characteristic of the product. One of these strains showing the strongest inhibition activity produces a bacteriocin, named Carnobacteriocin B2 that was involved in the antilisterial activity [105]. Three strains of bacteriocin producing *Carnobacterium* have been tested with the agar diffusion test method against a wide collection of *L. monocytogenes* (51 strains) isolated from seafood. All of the *Listeria* strains were sensitive. The inhibition was confirmed in co-culture with a mix of *L. monocytogenes* strains in sterile CSS [140]. One of these strains, *C. divergens* V41 showed its ability to maintain *L. monocytogenes* at the initial inoculating level of 20 CFU g-1 during 28 days of storage at 4°C and 8°C. The effect of this strain on sensory characteristics and physico-chemical parameters revealed that it did not spoiled the product [56].

A bacteriocinogenic strain of *L. sakei* isolated from CSS allowed a 4 log reduction of *Listeria innocua* after 14 days of storage at 4°C. A reduction of 2 log units after 24 h at 5°C was also demonstrated with that strain in CSS juice towards *L. monocytogenes* [141]. Mix of bacteriocin-producing LAB like *L. casei, L. plantarum* and *C. maltaromaticum* were successfully used to limit the growth of *L. innocua* in CSS [142]. *C. maltaromaticum* had no effect on the inhibition of the Gram positive spoilage bacteria *B. thermosphacta* in cooked shrimps [143]. The anti-listerial activity of 3 LAB strains used individually or as co-cultures was assayed on cold-smoked salmon artificially contaminated with *L. innocua* and stored under vacuum at 4°C [142]. The association of *L. casei* T3 and *L. plantarum* PE2 was the most effective, probably due to a competition mechanism against the pathogen. In their study Tomé et al. [144] have also selected a strain of *Enterococcus faecium* among five bacteriocinogenic LAB strains for its ability to induce a decrease of the population of *L. innocua* inoculated in CSS. However in these studies the inhibition activities were not confirmed on *L. monocytogenes.* For Matamoros et al. [145] two LAB strains, *Lactococcus piscium* EU2241 and *Leuconostoc gelidum* EU2247 were efficient to limit the growth of both pathogenic bacteria *L. monocytogenes* and *S. aureus* in a challenge test in cooked shrimp stored under VP from 2 to 3 log CFU g-1 units after 4 weeks at 8°C followed by 1 week at 20°C. The strain of *Leuconostoc* produced a bacteriocin-like compound but its activity was slight lower than the *Lactococcus* strain that was non-bacteriocinogenic. In another study, the application of *C. divergens* M35 towards *L. monocytogenes* in CSS resulted in a maximal decrease of 3.1 log CFU g-1 of the pathogenic bacteria after 21 days of storage at 4°C whereas a non bacteriocinogenic strain had no effect [119].

## **7. Conclusion and future prospective**

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

(3.5 μ/g), despite proteolytic degradation in the salmon.

spoiled the product [56].

this product, the combination of puried sakacin P and a live culture was found to be bactericidal against *L. monocytogenes*. The addition of sakacin P alone inhibited the growth of *L. monocytogenes* on this product for about 1 week [126]. Silva et al. [136] used a bacteriocinproducing *Carnobacterium* strain under a spray-dried format. This strain survived the process and retained antilisterial ability, although it lost activity against other Gram-positive targets such as *Staph. aureus.* Some authors have evaluated the antimicrobial activity of nisin combined with other bacteriocins. Bouttefroy & Milliere [137] tested combinations of nisin and curvaticin 13 produced by *L. curvatus* SB13 for preventing the regrowth of bacteriocinresistant cells of *L. monocytogenes*, finding that this combination induced a greater inhibitory effect than the use of a single bacteriocin. Aasen et al. [131] studied the interactions of the bacteriocins sakacin P and nisin with food constituents in cold-smoked salmon, chicken cold cuts, and raw chicken. They stated that owing to the amphiphilic nature of these peptides, they can be adsorbed to food macromolecules and undergo proteolytic degradation, which may limit their use as preservation agents. More than 80% of the added sakacin P and nisin were rapidly adsorbed by proteins in the food matrix that had not been heat-treated, less than 1% of the total activity remaining after 1 week in cold- smoked salmon. In heat-treated foods, they found that, bacteriocin activity was stable for more than 4 weeks. No important differences were observed between sakacin P and nisin, but less nisin was adsorbed by muscle proteins at low pH. The growth of *L. monocytogenes* was completely inhibited for at least 3 weeks in both chicken cold cuts and cold-smoked salmon by the addition of sakacin P

In the presence of the bacteriocinogenic strain *C. maltaromaticum* CS526 isolated from surimi, the population of *L. monocytogenes* in CSS decreased from 103 to 50 CFU g-1 after 7 days at 4°C [138]. This activity could be linked to the production of the bacteriocin piscicocin CS526, since a non-bacteriocin producing strain had a lower effect on the growth of the pathogenic bacteria [138, 139]. The growth of the protective *Carnobacterium* strains did not modify the sensory characteristic of the product. One of these strains showing the strongest inhibition activity produces a bacteriocin, named Carnobacteriocin B2 that was involved in the antilisterial activity [105]. Three strains of bacteriocin producing *Carnobacterium* have been tested with the agar diffusion test method against a wide collection of *L. monocytogenes* (51 strains) isolated from seafood. All of the *Listeria* strains were sensitive. The inhibition was confirmed in co-culture with a mix of *L. monocytogenes* strains in sterile CSS [140]. One of these strains, *C. divergens* V41 showed its ability to maintain *L. monocytogenes* at the initial inoculating level of 20 CFU g-1 during 28 days of storage at 4°C and 8°C. The effect of this strain on sensory characteristics and physico-chemical parameters revealed that it did not

A bacteriocinogenic strain of *L. sakei* isolated from CSS allowed a 4 log reduction of *Listeria innocua* after 14 days of storage at 4°C. A reduction of 2 log units after 24 h at 5°C was also demonstrated with that strain in CSS juice towards *L. monocytogenes* [141]. Mix of bacteriocin-producing LAB like *L. casei, L. plantarum* and *C. maltaromaticum* were successfully used to limit the growth of *L. innocua* in CSS [142]. *C. maltaromaticum* had no The presence of LAB in many processed seafood product is now well documented and the bio-protective potential of many strains and/or their bacteriocin has been highlighted in the last years. In situ production is readily cost-effective provided that the bacteriocin producers are technologically suitable. To date, only nisin and pediocin PA - 1 have been applied commercially in food applications where they are used to protect against spoilage and pathogenic organisms. However, other bacteriocins could be at least as effective for food processors as it is possible to apply them with hurdle approaches, particularly in light of consumer demands for minimally processed, safe, preservative - free foods. Control of pathogenic bacteria has widely focused on *L. monocytogenes* considered as the main risk in ready-to-eat seafood. However, in these minimally processed products, the new combination of hurdles can give selective advantages to enhance food safety and quality, particularly effective against other pathogenic bacteria like clostridia, vibrio or staphylococci. These goals can be facilitated through the incorporation of live bacteriocin producing strain(s) or through the use of bacteriocins as concentrated preparations, either through direct addition to the seafood or in an immobilized form on packaging as well as in conjunction with other factors such as high pressure or pulse electric fields, to achieve more effective preservation of foods. The great results obtained with protective culture, bacteriocins for improving safety and quality of seafood products clearly indicate that the application of LAB protective culture and/or their bacteriocins in seafood product can suggest several important benefits; 1) extended shelf life of seafood during storage time, 2) decrease the risk for transmission of foodborne pathogens in lightly preserved seafood products, 3) ameliorate the economic losses due to seafood spoilage, 4) reduce the application of chemical preservatives and drastic physical treatments such as heating, refrigeration, etc. causing better preservation nutritional quality of food, 5) good option for industry due to cost effective way and finally 6) a good response to consumer demands for minimally processed, safe, preservative - free foods. At present the new techniques and disciplines emerging in the post – genomic era, such as genomics, proteomics, metabolomics, and system biology, open new avenues for interpretation of biological data. In combination with classical and molecular techniques, these new methods will be invaluable in the rational optimization of LAB function in order to obtain safer traditional and new seafood products.

Lactic Acid Bacteria and Their Bacteriocins: A Promising Approach to Seafood Biopreservation 395

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