**4. The role of lactic acid bacteria in biopreservation technology**

### **4.1. Characterization and classification**

Lactic acid bacteria (LAB) encompass a heterogeneous group of microorganisms having as a common metabolic property the production of lactic acid as the majority end - product from the fermentation of carbohydrates [59]. LAB are Gram (+), usually nonmotile, non sporulating, catalase - negative, acid - tolerant, facultative anaerobic organisms and have less than 55 mol% G+C content in their DNA [60-62]. Except for a few species, LAB members are nonpathogenic organisms with a reputed generally recognized as safe status (GRAS). Taxonomic revisions of these genera and the description of new genera mean that LAB could, in their broad physiological definition, comprise around 20 genera [10]. However, from a practical, food-technology point of view, the following genera are considered the principal LAB: *Aerococcus*, *Carnobacterium*, *Enterococcus*, *Lactobacillus*, *Lactococcus*, *Leuconostoc*, *Oenococcus*, *Pediococcus*, *Streptococcus*, *Tetragenococcus*, *Vagococcus*, and *Weissella* [61]. The classification of lactic acid bacteria into different genera is largely based on morphology, mode of glucose fermentation, growth at different temperatures, configuration of the lactic acid produced, ability to grow at high salt concentrations, and acid or alkaline tolerance [62, 63]. An important characteristic used in the differentiation of the LAB genera is the mode of glucose fermentation under standard conditions. In this regard, the accepted definition is that given by Hommes and Vogel [64]: obligately homofermentative LAB are able to ferment hexoses almost exclusively to lactic acid by the Embden–Meyerhof–Parnas (EMP) pathway while pentoses and gluconate are not fermented as they lack phosphoketolase; facultatively heterofermentative LAB degrade hexoses to lactic acid by the EMP pathway and are also able to degrade pentoses and often gluconate as they possess both aldolase and phosphoketolase; finally, obligately heterofermentative degrade hexoses by the phosphogluconate pathway producing lactate, ethanol or acetic acid and carbon dioxide; moreover, pentoses are fermented by this pathway [62]. Several strains of groups 1 and 2 and some of the hetero fermentative group 3 are either used in fermented foods, but group 3 are also commonly associated with food spoilage. (For a more detailed discussion concerning the metabolic pathways, see [59].

### **4.2. Antimicrobial components from LAB**

### *4.2.1. Bacteriocins*

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

for other types of foods.

**4.1. Characterization and classification** 

concerning the metabolic pathways, see [59].

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

**4. The role of lactic acid bacteria in biopreservation technology** 

Lactic acid bacteria (LAB) encompass a heterogeneous group of microorganisms having as a common metabolic property the production of lactic acid as the majority end - product from the fermentation of carbohydrates [59]. LAB are Gram (+), usually nonmotile, non sporulating, catalase - negative, acid - tolerant, facultative anaerobic organisms and have less than 55 mol% G+C content in their DNA [60-62]. Except for a few species, LAB members are nonpathogenic organisms with a reputed generally recognized as safe status (GRAS). Taxonomic revisions of these genera and the description of new genera mean that LAB could, in their broad physiological definition, comprise around 20 genera [10]. However, from a practical, food-technology point of view, the following genera are considered the principal LAB: *Aerococcus*, *Carnobacterium*, *Enterococcus*, *Lactobacillus*, *Lactococcus*, *Leuconostoc*, *Oenococcus*, *Pediococcus*, *Streptococcus*, *Tetragenococcus*, *Vagococcus*, and *Weissella* [61]. The classification of lactic acid bacteria into different genera is largely based on morphology, mode of glucose fermentation, growth at different temperatures, configuration of the lactic acid produced, ability to grow at high salt concentrations, and acid or alkaline tolerance [62, 63]. An important characteristic used in the differentiation of the LAB genera is the mode of glucose fermentation under standard conditions. In this regard, the accepted definition is that given by Hommes and Vogel [64]: obligately homofermentative LAB are able to ferment hexoses almost exclusively to lactic acid by the Embden–Meyerhof–Parnas (EMP) pathway while pentoses and gluconate are not fermented as they lack phosphoketolase; facultatively heterofermentative LAB degrade hexoses to lactic acid by the EMP pathway and are also able to degrade pentoses and often gluconate as they possess both aldolase and phosphoketolase; finally, obligately heterofermentative degrade hexoses by the phosphogluconate pathway producing lactate, ethanol or acetic acid and carbon dioxide; moreover, pentoses are fermented by this pathway [62]. Several strains of groups 1 and 2 and some of the hetero fermentative group 3 are either used in fermented foods, but group 3 are also commonly associated with food spoilage. (For a more detailed discussion 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 linear peptide antimicrobials (class II).

### *4.2.2. Organic acid production*

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 slowing down metabolic activities, and in the case of Enterobacteriaceae such as *E. coli*  inhibiting growth at around pH 5.1.

Lactic Acid Bacteria and Their Bacteriocins: A Promising Approach to Seafood Biopreservation 389

[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

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

community of seafood.

**6. Application of LAB in seafood** 

### *4.2.3. Other antimicrobials of LAB*

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].
