**2. Bacterial hazards associated with fish and fish products**

From the viewpoint of microbiology, fish and related products are a risky foodstuff group. Pathogenic bacteria associated with seafood can be categorized into three general groups [14]: 1) Bacteria (indigenous bacteria) that belong to the natural microflora of fish (*Clostridium botulinum*, pathogenic *Vibrio* spp., *Aeromonas hydrophila*); 2) Enteric bacteria (non-indigenous bacteria) that are present due to faecal contamination (*Salmonella* spp., *Shigella* spp., pathogenic *Escherichia coli*, *Staphylococcus aureus*); and 3) bacterial contamination during processing, storage, or preparation for consumption (*Bacillus cereus*, *Listeria monocytogenes*, *Staphylococcus aureus*, *Clostridium perfringens, Clostridium botulinum, Salmonella* spp.).

*Vibrio parahaemolyticus* has been isolated from sea and estuary waters on all continents with elevated sea water temperatures. *V. parahaemolyticus* is frequently isolated from fish, molluscs, and crustaceans throughout the year in tropical climates and during the summer months in cold or temperate climates [15]. Fish food associated with illnesses due to consumption of *V. parahaemolyticus* includes fish-balls, fried mackerel (*Scomber scombrus*), tuna (*Thunnus thynnus*), and sardines (*Sardina pilchardus*). These products include both raw and undercooked fish products and cooked products that have been substantially recontaminated [9,15]. The most affected by the pathogens are Japan, Taiwan, and other Asian coastal regions, though cases of disease have been described in many countries and on many continents [9,16]. Cases of diseases caused by *V. parahaemolyticus* are occasional in Europe. During 20 years, only two cases of gastroenteritis were recorded in Denmark. The interest in this organism has been widened by the finding that similar organisms, *V. alginolyticus* and group of F *Vibrio* sp. also cause serious disease in humans [17]. *V. cholerae* is often transmitted by water but fish or fish products that have been in contact with contaminated water or faeces from infected persons also frequently serve as a source of infection [1,9,19]. The organism would be killed by cooking and recent cases of cholera in South America have been associated with the uncooked fish marinade seviche (*Cilus gilberti*) [18].

*E. coli* is a classic example of enteric bacteria causing gastroenteritis*. E. coli* including other coliforms and bacteria as *Staphylococcus* spp. and sometimes enterococci are commonly used as indices of hazardous conditions during processing of fish. Such organisms should not be present on fresh-caught fish [9,20,21]. The contamination fish derived food with pathogenic *E. coli* probably occurs during handling of fish and during the production process [20,22]. An outbreak of diarrhoeal illness caused by ingestion of food contaminated with enterotoxigenic *E. coli* was described in Japan [23]. The illness was strongly associated with eating tuna paste. Brazilian authors [24] isolated 18 enterotoxigenic strains of *E. coli* (ETEC) from 3 of 24 samples of fresh fish originating from Brazilian markets; 13 of them produced a thermolabile enterotoxin. Infection with verocytotoxin \_ producing strains of *E. coli* (VTEC) after ingestion of fish was recorded in Belgium [25]. An outbreak caused by salted salmon roe contaminated, probably during the production process, with enterohaemorrhagic *E. coli*  (EHEC) O157 occurred in Japan in 1998 [22]. The roe was stored frozen for 9 months but it appears that O157 could survive freezing and a high concentration of NaCl and retained its pathogenicity for humans [26].

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

*Salmonella* spp.).

[18].

are particularly interesting candidates for this technique [1,2,6,7]. Indeed, they are frequently naturally present in food products and are often strong competitors, by producing a wide range of antimicrobial metabolites such as organic acids, diacetyl, acetoin, hydrogen peroxide, reuterin, reutericyclin, antifungal peptides, and bacteriocins [8-10). Hence, the last two decades have seen intensive investigation on LAB and their metabolites

From the viewpoint of microbiology, fish and related products are a risky foodstuff group. Pathogenic bacteria associated with seafood can be categorized into three general groups [14]: 1) Bacteria (indigenous bacteria) that belong to the natural microflora of fish (*Clostridium botulinum*, pathogenic *Vibrio* spp., *Aeromonas hydrophila*); 2) Enteric bacteria (non-indigenous bacteria) that are present due to faecal contamination (*Salmonella* spp., *Shigella* spp., pathogenic *Escherichia coli*, *Staphylococcus aureus*); and 3) bacterial contamination during processing, storage, or preparation for consumption (*Bacillus cereus*, *Listeria monocytogenes*, *Staphylococcus aureus*, *Clostridium perfringens, Clostridium botulinum,* 

*Vibrio parahaemolyticus* has been isolated from sea and estuary waters on all continents with elevated sea water temperatures. *V. parahaemolyticus* is frequently isolated from fish, molluscs, and crustaceans throughout the year in tropical climates and during the summer months in cold or temperate climates [15]. Fish food associated with illnesses due to consumption of *V. parahaemolyticus* includes fish-balls, fried mackerel (*Scomber scombrus*), tuna (*Thunnus thynnus*), and sardines (*Sardina pilchardus*). These products include both raw and undercooked fish products and cooked products that have been substantially recontaminated [9,15]. The most affected by the pathogens are Japan, Taiwan, and other Asian coastal regions, though cases of disease have been described in many countries and on many continents [9,16]. Cases of diseases caused by *V. parahaemolyticus* are occasional in Europe. During 20 years, only two cases of gastroenteritis were recorded in Denmark. The interest in this organism has been widened by the finding that similar organisms, *V. alginolyticus* and group of F *Vibrio* sp. also cause serious disease in humans [17]. *V. cholerae* is often transmitted by water but fish or fish products that have been in contact with contaminated water or faeces from infected persons also frequently serve as a source of infection [1,9,19]. The organism would be killed by cooking and recent cases of cholera in South America have been associated with the uncooked fish marinade seviche (*Cilus gilberti*)

*E. coli* is a classic example of enteric bacteria causing gastroenteritis*. E. coli* including other coliforms and bacteria as *Staphylococcus* spp. and sometimes enterococci are commonly used as indices of hazardous conditions during processing of fish. Such organisms should not be present on fresh-caught fish [9,20,21]. The contamination fish derived food with pathogenic *E. coli* probably occurs during handling of fish and during the production process [20,22]. An outbreak of diarrhoeal illness caused by ingestion of food contaminated with

to discover new LAB strains that can be used in food preservation [1,7,11-13].

**2. Bacterial hazards associated with fish and fish products** 

*Aeromonas* spp. has been recognized as potential foodborne pathogens for more than 20 years. Aeromonads are ubiquitous in fresh water, fish and shellfish and also in meats and fresh vegetables [27]. The epidemiological results so far are, however, very questionable. The organism is very frequently present in many food products, including raw vegetables, and very rarely has a case been reported. Up to 8.1% of cases of acute enteric diseases in 458 patients in Russia were caused by *Aeromonas* spp. [28]. In this study, *Aeromonas* spp. isolates with the same pathogenicity factors were isolated from river water in the Volga Delta, from fish, raw meat, and from patients with diarrhoea. Most *Aeromonas* spp. isolates are psychrotrophic and can grow at refrigerator temperatures [29]. This could increase the hazard of food contamination, particularly where there is a possibility of crosscontamination with ready-to-eat food products.

*Salmonella* has been isolated from fish and fishery product, though it is not psychrotrophic or indigenous to the aquatic environment [30]. The relationship between fish and *Salmonella*  has been described by several scientists; some believe that fish are possible carriers of *Salmonella* which are harbored in their intestines for relatively short periods of time and some believe that fish get actively infected by *Salmonella* [31]. Most outbreaks of food poisoning associated with fish derive from the consumption of raw or insufficiently heat treated fish and cross-contamination during processing and the U.S. Food and Drug Administration's (FDA) data showed that *Salmonella* was the most common contaminant of fish and fishery products [31]. The highest *Salmonella* incidence in fishery products was determined in Central Pacific and African countries while it was lower in Europe and including Russia, and North America [32]. The most common serovar found in the world was *S.* sub Weltvreden [30, 31]. In seafood the commonest serotype encountered was *S.* sub Worthington followed by *S.* sub Weltevreden.

Enterotoxins produced by *Staphylococcus aureus* are another serious cause of gastroenteritis after consumption of fish and related products. In 3 of 10 samples of fresh fish, higher counts of *Staph. aureus* were detected than permitted by Brazilian legislation [20, 33]. In the southern area of Brazil, *Staph. aureus* was isolated from 20% of 175 examined samples of fresh fish and fish fillets (*Cynoscion leiarchus*). *Staph. aureus* has also been detected during the process of drying and subsequent smoking of eels in Alaska in 1993 [34]. During the process, *S. aureus* populations increased to more than 105 CFU g-1 of the analyzed sample, after 2 to 3 days of processing. Subsequent laboratory studies showed that a pellicle (a dried skin-like surface) formed rapidly on the strips when there was rapid air circulation in the smokehouse and that bacteria embedded in/under the pellicle were able to grow even when heavy smoke deposition occurred.

Lactic Acid Bacteria and Their Bacteriocins: A Promising Approach to Seafood Biopreservation 385

viscera allowed the bacterium to multiply and to produce toxin. *C. perfringens*, an important cause of both food poisoning and non-food-borne diarrhoeas in humans, was found in a number of fish owing to contamination with sewage, which is the main source of this

Seafood products are known to be especially susceptible to both microbiological and biochemical spoilage pathways. The development of effective processing treatments to extend the shelf life of fresh fish products is a must [2]. Additionally, the consumers' demand for high-quality and minimally processed seafood has recently captivated great attention [5, 9]. However, an increase in foodborne illness outbreaks is concomitant with the increase in consumer demand for less processed foods [1]. These trends highlight the importance of studying new microbial stress factors to extend the shelf-life of foods. Until now, approaches to reduce the risk of outbreaks of food poisoning have relied on the search for addition of more efficient chemical preservatives or on the application of more drastic physical treatments such as heating, refrigeration, high hydrostatic pressure (HHP), ionising radiation, pulsed-light, ozone, ultrasound, etc [1,5,50]. In spite of some possible advantage, these types of treatments have many drawbacks and limitation in seafood products: the proven toxicity of many of the commonest chemical preservatives (e.g. nitrites) (3), the alteration of the organoleptic and nutritional properties of seafood by physical treatments due to their delicate nature (e.g. freezing damage, discolouration in case of HHP and ionising radiation) [50,51] and especially recent consumer trends in purchasing and consumption, with demands for healthy seafood products that have been subjected to less extreme treatments (less heat and chill damage), with lower levels of salts, fats, acids, and sugars and/or the complete or the partial removal of chemically synthesized additives [1,2,7]. To harmonize consumer demands with the necessary safety standards, traditional means of controlling microbial spoilage and safety hazards in seafood are being replaced by an alternative solution that is gaining more and more attention: "biopreservation technology" [2,9,13,52,53]. It consists in inoculating food with microorganisms, or their metabolites, selected for their antibacterial properties and may be an efficient way of extending shelf life and food safety through the inhibition of spoilage and pathogenic bacteria without altering the nutritional quality of raw materials and food products [54, 55].

Lactic acid bacteria (LAB) possess a major potential for use in biopreservation because they are safe to consume, and during storage they naturally dominate the microbiota of many foods. Certain LAB species and strains isolated from seafood have been shown to exert strong antagonistic activity against spoilage and pathogenic microorganisms such as *Listeria, Clostridium, Staphylococcus*, and *Bacillus* spp [56-58]. The antagonistic and inhibitory properties of LAB are due to the competition for nutrients and the production of one or more antimicrobially active metabolites such as organic acids (lactic and acetic acid), hydrogen peroxide, and antimicrobial peptides (bacteriocins) [10]. Certain LAB are able to grow at refrigeration temperatures and are tolerant to modified-atmosphere packaging, low

organism [21].

**3. Biopreservation** 

In ready-to-eat products, cooking, preservation ingredients, and storage atmosphere inhibit the Gram-negative organisms, resulting in a longer shelf life. Such conditions favor the growth of psychotropic pathogens such as *Listeria monocytogenes*, allowing them to grow to dangerous levels [9,35,36]. *L. monocytogenes* is a serious threat to consumer health and safety and has been implicated in several deadly outbreaks around the world [1,2]. This organism is halotolerant (up to 28% w/v for short periods), resistant to freezing temperatures, can grow and multiply during refrigeration, where other competing organisms cannot, and is able to survive at low water activity (aw) [9,14,37]. *L. monocytogenes* is widely distributed in the general environment including fresh water, coastal water and live fish from these areas. Contamination or recontamination of seafood may also take place during processing [37-39]. Moreover, *L. monocytogenes* is a psychrotrophic pathogen with the ability to grow from under 0 to 45°C [40]. This ability to grow at storage temperatures means that this bacterium is the main hazard in this kind of product. The pathogenic bacteria *L. monocytogenes* may grow on fresh seafood. *Listeria* has been found in farmed rainbow trout [41]. The outbreak of listeriosis related to vacuum packed gravad and cold-smoked fish was described in at least eight human cases for 11 months in Sweden [42]. Cold-smoked and gravad rainbow trout (*Oncorhynchus mykiss*) and salmon (*Salmo salar*) have been focused on during recent years as potential sources of infection with *L. monocytogenes* and there are several report on isolation of this food borne pathogen from fish-processing plants environments [14,37,39,43-45]. Seafood treatment is necessary to prevent food-borne illness. However, the pervasive nature of *L. monocytogenes* makes it difficult for processors to fully eliminate the organism from the environment.

Development of new-generation foods, which are mildly processed, contain few or no preservatives, are packaged in vacuum or modified atmospheres to ensure long shelf life and rely primarily on refrigeration for preservation, has raised concerns of potential increases in botulism risk caused by psychrotrophic nonproteolytic group II *Clostridium botulinum* [46]. An average of 450 outbreaks of foodborne botulism with 930 cases have been reported annually worldwide [47]. The main habitat of clostridia is the soil but they are also found in sewage, rivers, lakes, sea water, fresh meat, and fish [48,49]. Most critical are the hygienic conditions for handling the product after smoking. There is a risk of botulism due to the growth of *C. botulinum* type E in smoked fish. The bacterium becomes a hazard when processing practices are insufficient to eliminate botulinal spores from raw fish, particularly improper thermal processing [21]. The growth of *C. botulinum* and toxin production then depends on appropriate conditions in food before eating: the temperature, oxygen level, water activity, pH, the presence of preservatives, and competing microflora [21]. A problem with *C. botulinum* has been encountered with some traditional fermented fish products. These rely on a combination of salt and reduced pH for their safety. If the product has insufficient salt, or fails to achieve a rapid pH drop to below 4.5, *C. botulinum* can grow. There was no evidence that the fish had been mishandled, but a low salt environment in the viscera allowed the bacterium to multiply and to produce toxin. *C. perfringens*, an important cause of both food poisoning and non-food-borne diarrhoeas in humans, was found in a number of fish owing to contamination with sewage, which is the main source of this organism [21].

### **3. Biopreservation**

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

heavy smoke deposition occurred.

environment.

surface) formed rapidly on the strips when there was rapid air circulation in the smokehouse and that bacteria embedded in/under the pellicle were able to grow even when

In ready-to-eat products, cooking, preservation ingredients, and storage atmosphere inhibit the Gram-negative organisms, resulting in a longer shelf life. Such conditions favor the growth of psychotropic pathogens such as *Listeria monocytogenes*, allowing them to grow to dangerous levels [9,35,36]. *L. monocytogenes* is a serious threat to consumer health and safety and has been implicated in several deadly outbreaks around the world [1,2]. This organism is halotolerant (up to 28% w/v for short periods), resistant to freezing temperatures, can grow and multiply during refrigeration, where other competing organisms cannot, and is able to survive at low water activity (aw) [9,14,37]. *L. monocytogenes* is widely distributed in the general environment including fresh water, coastal water and live fish from these areas. Contamination or recontamination of seafood may also take place during processing [37-39]. Moreover, *L. monocytogenes* is a psychrotrophic pathogen with the ability to grow from under 0 to 45°C [40]. This ability to grow at storage temperatures means that this bacterium is the main hazard in this kind of product. The pathogenic bacteria *L. monocytogenes* may grow on fresh seafood. *Listeria* has been found in farmed rainbow trout [41]. The outbreak of listeriosis related to vacuum packed gravad and cold-smoked fish was described in at least eight human cases for 11 months in Sweden [42]. Cold-smoked and gravad rainbow trout (*Oncorhynchus mykiss*) and salmon (*Salmo salar*) have been focused on during recent years as potential sources of infection with *L. monocytogenes* and there are several report on isolation of this food borne pathogen from fish-processing plants environments [14,37,39,43-45]. Seafood treatment is necessary to prevent food-borne illness. However, the pervasive nature of *L. monocytogenes* makes it difficult for processors to fully eliminate the organism from the

Development of new-generation foods, which are mildly processed, contain few or no preservatives, are packaged in vacuum or modified atmospheres to ensure long shelf life and rely primarily on refrigeration for preservation, has raised concerns of potential increases in botulism risk caused by psychrotrophic nonproteolytic group II *Clostridium botulinum* [46]. An average of 450 outbreaks of foodborne botulism with 930 cases have been reported annually worldwide [47]. The main habitat of clostridia is the soil but they are also found in sewage, rivers, lakes, sea water, fresh meat, and fish [48,49]. Most critical are the hygienic conditions for handling the product after smoking. There is a risk of botulism due to the growth of *C. botulinum* type E in smoked fish. The bacterium becomes a hazard when processing practices are insufficient to eliminate botulinal spores from raw fish, particularly improper thermal processing [21]. The growth of *C. botulinum* and toxin production then depends on appropriate conditions in food before eating: the temperature, oxygen level, water activity, pH, the presence of preservatives, and competing microflora [21]. A problem with *C. botulinum* has been encountered with some traditional fermented fish products. These rely on a combination of salt and reduced pH for their safety. If the product has insufficient salt, or fails to achieve a rapid pH drop to below 4.5, *C. botulinum* can grow. There was no evidence that the fish had been mishandled, but a low salt environment in the Seafood products are known to be especially susceptible to both microbiological and biochemical spoilage pathways. The development of effective processing treatments to extend the shelf life of fresh fish products is a must [2]. Additionally, the consumers' demand for high-quality and minimally processed seafood has recently captivated great attention [5, 9]. However, an increase in foodborne illness outbreaks is concomitant with the increase in consumer demand for less processed foods [1]. These trends highlight the importance of studying new microbial stress factors to extend the shelf-life of foods. Until now, approaches to reduce the risk of outbreaks of food poisoning have relied on the search for addition of more efficient chemical preservatives or on the application of more drastic physical treatments such as heating, refrigeration, high hydrostatic pressure (HHP), ionising radiation, pulsed-light, ozone, ultrasound, etc [1,5,50]. In spite of some possible advantage, these types of treatments have many drawbacks and limitation in seafood products: the proven toxicity of many of the commonest chemical preservatives (e.g. nitrites) (3), the alteration of the organoleptic and nutritional properties of seafood by physical treatments due to their delicate nature (e.g. freezing damage, discolouration in case of HHP and ionising radiation) [50,51] and especially recent consumer trends in purchasing and consumption, with demands for healthy seafood products that have been subjected to less extreme treatments (less heat and chill damage), with lower levels of salts, fats, acids, and sugars and/or the complete or the partial removal of chemically synthesized additives [1,2,7]. To harmonize consumer demands with the necessary safety standards, traditional means of controlling microbial spoilage and safety hazards in seafood are being replaced by an alternative solution that is gaining more and more attention: "biopreservation technology" [2,9,13,52,53]. It consists in inoculating food with microorganisms, or their metabolites, selected for their antibacterial properties and may be an efficient way of extending shelf life and food safety through the inhibition of spoilage and pathogenic bacteria without altering the nutritional quality of raw materials and food products [54, 55].

Lactic acid bacteria (LAB) possess a major potential for use in biopreservation because they are safe to consume, and during storage they naturally dominate the microbiota of many foods. Certain LAB species and strains isolated from seafood have been shown to exert strong antagonistic activity against spoilage and pathogenic microorganisms such as *Listeria, Clostridium, Staphylococcus*, and *Bacillus* spp [56-58]. The antagonistic and inhibitory properties of LAB are due to the competition for nutrients and the production of one or more antimicrobially active metabolites such as organic acids (lactic and acetic acid), hydrogen peroxide, and antimicrobial peptides (bacteriocins) [10]. Certain LAB are able to grow at refrigeration temperatures and are tolerant to modified-atmosphere packaging, low pH, high salt concentrations, and the presence of certain additives such as lactic acid, acetic acid, and ethanol. Because of these benefits, LAB can be used as protective cultures to restrict the growth of undesired organisms such as certain spoilage and pathogenic bacteria, with the subsequent benefits in terms of food safety [9,10,58]. Moreover, these microorganisms may have additional functional properties and, in some circumstances, they can be beneficial for the consumers [6]. LAB represent the microbial group most commonly used as protective cultures, as they are present in all fermented foods and have a long history of safe use [8]. Safety for the consumers is an aspect of great importance, in particular for some seafood products which are not cooked before consumptions, but also for other types of foods.

Lactic Acid Bacteria and Their Bacteriocins: A Promising Approach to Seafood Biopreservation 387

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) [1,2,7]. It has been estimated that between 30% and 99% of all bacteria and archaea produce bacteriocins; their production by LAB is very significant from the point of view of their potential applications in food systems and thus, unsurprisingly, these have been most extensively investigated [6,10,12,60,65,66]. It has been noted that the activity of bacteriocins is frequently directed against bacteria that are related to the bacteriocin - producing strain or against bacteria found in similar environments [67]. It has also been noted that some bacteriocins can also play a role in cell signaling. Microorganisms that produce bacteriocins also possess immunity mechanisms to confer self - protection, that is, to protect bacteriocin producers from committing "suicide" [10,68,69]. Besides concern about antibiotic resistance, increasing consumer awareness of potential health risks associated with chemical preservatives has increased interest in bacteriocins. Bacteriocins are naturally produced so they are more easily accepted by consumers [54]. Bacteriocins are usually classified combining various criteria. The main ones being the producer bacterial family, their molecular weight and finally their amino acid sequence homologies and/or gene cluster organization [59,70]. Based on a relatively recent approach [69,71,72] bacteriocins produced by LAB have been categorized into two major classes: the lanthionine - containing bacteriocins or lantibiotics (class I) and the largely unmodified

An important role of meat LAB starter cultures is the rapid production of organic acids; this inhibits the growth of unwanted flora and enhances product safety and shelf life. The types and levels of organic acids produced during the fermentation process depend on the LAB strains present, the culture composition, and the growth conditions [74]. Fermentation of the carbohydrates, glucose, glycogen, glucose-6-phosphate and small amounts of ribose, in meat and meat products, produces organic acids by glycolysis (Embden-Meyerhof Parnas pathway, EMP pathway) or the Hexose Monophosphate, HMP pathway. L (+) lactic acid is more inhibitory than its D (-) counterpart [68]. The antimicrobial effect of organic acids lies in the reduction of pH, and in the action of undissociated acid molecules [75]. It has been proposed that low external pH causes acidification of the cytoplasm. The lipophilic nature of the undissociated acid allows it to diffuse across the cell membrane collapsing the electrochemical proton gradient. Alternatively, cell membrane permeability may be affected, disrupting substrate transport systems [72]. The LAB in particular are able to reduce the pH to levels where putrefactive (e.g. clostridia and pseudomonads), pathogenic (e.g. *Salmonella*  s and *Listeria* spp.) and toxinogenic bacteria (*Staphylococcus aureus*. *Bacillus cereus*, *Clostridium botulinum*) will be either inhibited or killed [7]. Also, the undissociated acid, on account of its fat solubility, will diffuse into the bacterial cell, thereby reducing the intracellular pH and

**4.2. Antimicrobial components from LAB** 

linear peptide antimicrobials (class II).

*4.2.2. Organic acid production* 

*4.2.1. Bacteriocins* 
