Leuconostoc Review Denis Hemme

ARTIC AR TICLE LE IN PR PRESS ESS

International Dairy Journal 14 (2004) 467–494

Review

Leuconostoc, Leuconostoc, characteristics, use in dairy technology and prospects in functional foods Denis Hemme, Catherine Foucaud-Scheunemann* ! etique ! ! 78352 Jouy en Josas Cedex, France Institut National de la Recherche Agronomique, Unit e! de Recherches Laiti eres e" res et G en Appliquee,

Received 14 March 2003; accepted 2 October 2003 This review is dedicated to the memory of Charles Divie` s, professor at ENSBANA, Universite` de Bourgogne, Dijon, France who passed away January 8th, 2003

Abstract

Leuconostoc, relative to their habitat, taxonomy, metabolism, and This review presents up to date comprehensive data about Leuconostoc, genetics, their implications in health and safety, and their present and potential use in dairy technology and functional foods. r 2003 Elsevier Ltd. All rights reserved. Keywords: Leuconostoc Leuconostoc;; Review; Review; Cheese; Cheese; Dairy Dairy products products;; Habitat; Habitat; Classific Classificatio ation; n; Taxonom Taxonomy; y; Bacterio Bacteriopha phages; ges; Technolo Technology; gy; Bacterio Bacteriocins cins;; Metabolism; Plasmids; Genetics; Polysaccharides; Clinical occurrence; Infection

Contents

1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2. Habi Habita tatt of Leuconostoc of Leuconostoc . . . . . . . . . . . . 2.1. Presence in various niches . . . . . . . 2.2. 2.2. Pres Presen ence ce in ferm fermen ente ted d prod produc ucts ts,, gene genera rall 2.3. Presence in dairy products . . . . . . .

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Health, disease and safety . . . . . . . 3.1. Infection and opportunism . . . . 3.2. Antibiotic resistance . . . . . . . 3.3. Health-related metabolic activities

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Characterization and taxonomy . . . . . . . . . . . 4.1. 4.1. Iden Identi tific ficat atiion, on, char charac acte terrisat isatiion and and biod biodiv iveersity sity 4.2. Recent evolution and up to date taxonomy . . 4.3. Comments about methods of comparison . . .

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5. Grow Growth th of Leuconostoc of Leuconostoc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. 5.1. Cult Cultiv ivat atio ion, n, main mainte tena nanc ncee and and pres preser erva vati tion on of cult cultur ures es . . . . . . . . . . . . 5.1.1. Media for isolation and enumeration . . . . . . . . . . . . . . . . 5.1.2. Preservation of cultures . . . . . . . . . . . . . . . . . . . . . . . 5.2. Industrial production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. 5.3. Involv Involvem ement ent of Leuconostoc of Leuconostoc in microbial interaction . . . . . . . . . . . . . 5.3.1. Co-aggregation reactions . . . . . . . . . . . . . . . . . . . . . . 5.3.2.. Growth 5.3.2 Growth and and metabo metabolism lism of Leuconostoc in mixed strain cultures . .

*Corresponding author. Tel.: +33-1-3465-2091; fax: + 33-1-3465-2065. E-mail address: catherine.foucaud@j [email protected] ouy.inra.fr a.fr (C. Foucaud-Scheuneman Foucaud-Scheunemann). n).

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D. Hemme, C. Foucaud-Scheunemann / International Dairy Journal 14 (2004) 467–494

5.3. 5.3.3. 3. Meta Metabo boli licc end end prod produc ucts ts and and inte interf rfer eren ence ce with with micr microb obia iall grow growth th . . . . . . . . . . . . . . 5.3.4.. Bacteriop 5.3.4 Bacteriophages hages of dairy dairy Leuconostoc . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leuconostoc . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.5.. Bacterioc 5.3.5 Bacteriocins ins produ produced ced by dairy dairy Leuconostoc.

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6. Surv Surviv ival al of Leuconostoc of Leuconostoc to stress conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Metabo Metabolis lism m of Leuconostoc . . . . . . . . . 7.1. Carbohydrate utilisation . . . . . . . 7.1.1. Sugar transport . . . . . . . 7.1.2. Sugar catabolism . . . . . . . 7.2. Organic acid metabolism . . . . . . . 7.3. Nitrogenous compounds metabolism . 7.3.1. Amino acid requirements . . 7.3.2. The proteolytic system . . . . 7.4. Metabolism in the presence of oxygen 7.5. Other metabolic considerations . . . .

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9. Role Roless of Leuconostoc of Leuconostoc in technology . . . . . . . . . . . . . 9.1. Roles in dairy technology . . . . . . . . . . . . . . . 9.1.1. Openness . . . . . . . . . . . . . . . . . . . . 9.1.2. Aroma production . . . . . . . . . . . . . . . 9.1.3.. Deliberat 9.1.3 Deliberatee additi addition on of Leuconostoc as NSLAB 9.2. Roles in functional foods . . . . . . . . . . . . . . . . 9.2.1. Leuconostoc as potential probiotics . . . . . . 9.2.2. Production of polysaccharides . . . . . . . . . 9.2.3. Production of mannitol . . . . . . . . . . . . 9.2.4.. Hydrolys 9.2.4 Hydrolysis is of a-galactosides . . . . . . . . . . 9.2.5. Production of vitamins . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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8. Gene Geneti tics cs of Leuconostoc of Leuconostoc . . . . . . . . 8.1. Plasmid biology . . . . . . . . . 8.2. Cloning vectors and gene transfer 8.3. Gene cloning and expression . . . 8.4. Genomics . . . . . . . . . . . . .

Conclusion and prospects . . . . 10.1. Identification and ecology . 10.2. Health, safety and benefits 10.3. Technological implications

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1. Introduction Introduction

Strains belonging to the genus Leuconostoc are Grampositi positive ve lactic lactic acid acid bacteri bacteria a (LAB) (LAB) of econom economic ic imimportance, related to numerous positive aspects: *

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Fermentation of foodstuffs (sauerkraut, pickles, meat products, etc.). Production of gas (CO2) in cheeses presenting openness ( in particular Blue-veined cheeses). Production of flavour compounds in multiple dairy products. In situ production of dextran in saccharose containing (dairy) products, or as high value polymers for industrial or clinical use, or as biological auxiliary in the oil industry. Potential roles in functional foods.

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Leuconostoc strains are also linked to a few negative aspects aspects includ including ing spoila spoilage ge in the sugar sugar cane cane indust industry ry (Day Day,, 199 1992 2) and and food food prod produc ucts ts (Ennah Ennahar, ar, Cai, & Fujita Fuj ita,, 200 2003 3; Ly Lyhs hs,, 20 2002 02;; Susil Susiluoto, uoto, Kork Korkeala, eala, & Bjorkroth, 2003) 2003) by formation of slime, and safety as they they have have been been identi identified fied in human human clinic clinical al isolat isolates es (Secti (Section on 3). Howeve However, r, the history history of human human exposu exposure re and and cons consum umpt ptio ion n has has led led to the the conc conclu lusi sion on that that Leuconostoc is a microo microorga rganis nism m genera generally lly regard regarded ed as safe. In dairy technology, the importance of Leuconostoc strains is widely recognized, although their physiology and genetics are less developed than those of Lactococof Lactococcus. They are often present in dairy starter cultures and also also in the the dair dairy y envi enviro ronm nmen entt and and thus thus coul could d be considered as non-starter lactic acid bacteria (NSLAB) in the the same same way way as meso mesoph phil ilic ic lact lactob obac acil illi li (Cogan,

ARTICLE IN PRESS D. Hemme, C. Foucaud-Scheunemann / International Dairy Journal 14 (2004) 467–494

2002). Their role in the formation of aroma and texture of certain dairy products is essential. This review will synthesize data concerning Leuconostoc species, which play a role in the dairy field, excluding in particular information relative to Oenococcus oeni and Weissella paramesenteroides. Main comprehensive reviews dealing with Leuconostoc were either ancient (Devoyod & Poullain, 1988) or partial with emphasis on taxonomy (Thunell, 1995), metabolism (Cogan & Jordan, 1994), biotechnology (Dessart & Steenson, 1995), bacteriocins (Stiles, 1994) or dairy technology (Vedamuthu, 1994) or are included in extensive review on LAB (Stiles & Holzapfel, 1997; McSweeney & Sousa, 2000; Carr, Chill, & Maida, 2002) or in a book chapter (Bjorkroth & Holzapfel, 2003). To avoid extensive list of references, those cited in these reviews or in original articles are omitted and the readers might refer to references given herein.

2. Habitat of Leuconostoc

2.1. Presence in various niches Leuconostoc strains present in many environments originate from green vegetation and roots that constitute their natural ecological niche (Mundt, 1970), although their population is often small (less than 1%) compared with that of aerobic bacteria and yeasts . (Buckenhuskes, 1993). From this natural habitat, they can easily propagate in various niches including plant materials such as vegetables and silage (Ennahar et al., 2003) and fermentation food products from various raw materials (Table 1). Their presence in milk and consequently in dairy products, is due to contamination during milking and (or) manufacturing, enhanced by their peculiarity of surviving for a long time on material surfaces such as wooden ‘‘gerle’’ and other moulds (Devoyod & Poullain, 1988) or through the pasteuriser (Martley & Crow, 1993). Leuconostoc is not usually considered as part of the human flora although strains have been isolated from human faeces, vaginal samples and breast milk samples (Auge, Donnio, Le De! aut, & Avril, 1987; Green, Wadowsky, & Barbadora, 1990; Heikkila. , & Saris, 2003; dal Bello, Walter, Hammes, & Hertel, 2003) and also in internal fluids (blood, urine and cerebrospinal fluid) of disabled patients (Farrow, Facklam, & Collins, 1989). They have also been isolated from the microflora of cattle (Brashears, Jaroni, & Trimble, 2003), fish (Ringo & Gatesoupe, 1998), insects (Ohkuma & Kudo, 1998; Reeson, Jankovic, Kasper, Rogers, & Austin, 2003) and from other environments such as treated bauxite residues (Hamdy & Williams, 2001). Leuconostoc was one of the first bacterial groups studied for their causative role in commercial losses in

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sugar industry (Day, 1992). They have also been associated with spoilage of fish (Lyhs, 2002) and meat products (Bjorkroth et al., 2000; Anifantaki, Metaxopoulos, Kammenou, Drosinos, & Vlassi, 2002; Hamasaki, Ayaki, Fuchu, Sugiyama, & Morita, 2003). 2.2. Presence in fermented products, general considerations LAB including Leuconostoc, play an important role in the fermentation of various products including foods (Table 1). The source of the microbial flora can be either the raw material as for the production of raw milk cheeses, sauerkraut and some fermented sausages or a commercial starter cultures. The first procedure is often in use for propagation of sourdough for private use, and for the production of some artisanal cheeses where the inoculum is maintained by keeping a sample from the previous production, the so-called ‘‘old-young technique’’ (Limsowtin, Powell, & Parente, 1996). During fermentation, the end products of carbohydrate metabolism contribute not only to the preservation upon acidification but also to the flavour and texture of the product. Fermentation may also increase the nutritional quality of food by increasing its digestibility, as in the fermentation of milk to cheese, or by reducing its toxicity (Gueguen, Chemardin, Labrot, Arnaud, & Galzy, 1997). 2.3. Presence in dairy products Ln. mesenteroides subsp. cremoris or Ln. lactis strains are of classical use in butter and cream production and some fermented fresh dairy products (Vedamuthu, 1994; Section 9.1). The presence of Leuconostoc in numerous cheese varieties made without addition of Leuconostoc starter is regular, in particular in raw milk cheeses. In an extensive study, among 4379 isolates from 35 different European dairy products including 24 artisanal cheeses, 10% of the strains were Leuconostoc (Cogan et al., 1997). This level has to be compared to the levels of lactobacilli (mesophile, 12% and thermophile, 14%) and enterococci (17%). These results confirm previous data indicating the presence of Leuconostoc in the majority of raw milk French cheeses, and other European cheeses (Devoyod & Poullain, 1988). Numerous studies are still devoted to the ecology of artisanal cheeses, i.e. nature and evolution of the main microbial groups during manufacture. The studies published in the last 5 years concerned mostly raw milk cheeses from South-European countries, and also France, Great-Britain, Sweden, and India and relied progressively on new DNA-based methods (Estepar, del Mar Sanchez, Alonso, & Mayo, 1999; Morea, Baruzzi, & Cocconcelli, 1999; Lopez-Diaz, Alonso, Roman,

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Table 1 Fermented foods that involve Leuconostoc a Products

Foodstuff

Raw material

Country

Microorganisms

Dairy

Butter and cream Cheeses Fermented milks (amasi, maziwa lala, laban, filmjolk, kefir, pindidam, smetanka, etc)

Milk Milk Milk

International International Europe

LAB LAB, yeasts, mould LAB, yeasts

Africa Asia Meat

Fish Cereal

Vegetable

Sausages

Meat

Salami Sauce foods (belacham, chinchaluk, pekasam, som-fak, etc) Beverages (beer, boza, bushera, idli, dadih, jangsu, ogi, pozol, sobia, etc) Dough and starchy accompaniments (bread, flour, mawe, puto, trahanas, etc) Sauce foods (tsauco, etc) Sauerkraut Pickles, kimchi, sayur-asin

Dough and starchy accompaniments (agbelima, flour, fufu, sapal, etc)

Fruit

LAB, yeasts, moulds

Meat

Europe Southeast Asia Europe

Fish, schrimp

Southeast Asia

LAB

Malt, maize, corn, rice, millet

International

LAB, yeasts, moulds

Maize, rice, sorghum, tef

Europe, Africa, Southeast Asia

LAB, yeasts

Rice, soybeans

Southeast Asia

LAB

Cabbage Olives, beetroot, cabbage, carrot, cucumber, sweetpepper Cassava, taro

International International

LAB LAB

Africa,

LAB

LAB

Cocoa

New Guinea South America Central Africa

Coffee

South America Central Africa

Juices

South America International

LAB

South East Asia Africa

LAB LAB

Tempoyak Kocho

Durian fruit Ensete ventricosa

Yeast, acetic acid bacteria, LAB LAB, yeasts, Enterobacteriaceae,

a

Compiled from Arunga (1992), Buckenh uskes . (1993), Thunell (1995), Caplice and Fitzgerald, 1999, Steinkraus (2002), Avallone, Guyot, Brillouet, Olguin, and Guiraud (2001) , Mogensen et al. (2002), Hansen (2002), Bjorkroth and Holzapfel (2003).

Garcia Lopez, & Moreno, 2000; Tamang et al., 2000; Casalta et al., 2001; Garcia Fontan, Franco, Prieto, Tornadijo, & Carballo, 2001; Menendez, Godinez, Centeno, & Rodriguez-Otero, 2001; Alonso-Calleja, Carballo, Capita, Bernardo, & Garcia-Lopez, 2002; Mas et al. 2002; Ostlie, Eliassen, & Skeie, 2002; Arenas, Gonzalez, Bernardo, Fresno, & Tornadijo, 2003; Duthoit, Godon, & Montel, 2003; Ercolini, Hill, & Dodd, 2003; Herreros, Fresno, Gonzalez Prieto, & Tornadijo, 2003; Manolopoulou et al., 2003; Tavaria & Malcata, 2003). Leuconostoc was not detected as NSLAB in American (Swearingen, O’Sullivan, & Warthesen, 2001) and Irish (Fitzsimons, Cogan, Condon, & Beresford, 1999) quality Cheddar cheeses whereas it has been detected in cheeses of UK origin (Williams, Choi, & Banks, 2002)

or with split defect (Peterson & Marshall, 1990) using phenotypic tools and (or) DNA-based methods. The recovery of Leuconostoc might rely on the origin (studies of Irish cheeses concerned various manufacturers over different periods in Ireland in contrast to other work dedicated to one factory), the maturity status, the manufacturing practices and also the quality of Cheddar cheeses. Hard cheeses did not contain Leuconostoc strains, mainly due to their manufacturing process. Leuconostoc is present in a large variety of fermented milks (Table 1). It is also one of the components of the kefir grain contributing, slightly compared with yeasts, to the production of ethanol and acetate, which are characteristic of this product (Robinson, Tamime, & Wszolek, 2002).


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growth, which allows them to multiply and cause disease (Auge et al., 1987).

3. Health, disease and safety

A number of reports associate LAB, including Leuconostoc, with clinical infections. Attention was also devoted to the enzymatic activities implicated in the production of potential deleterious substances. 3.1. Infection and opportunism Originally considered to be clinically insignificant, probably as it was not specifically detected, Leuconostoc is now slightly emerging as opportunistic pathogen until the first report of bacteraemia caused by Leuconostoc came out in 1985 (Dhodapkar & Henry, 1996). Less than 100 cases of Leuconostoc infections have been reported since, mostly involving adults with compromised immunity or a wide spectrum of underlying diseases although infections have also been documented in few healthy patients. Predisposing factors also include the use of enteral or central venous catheter and other invasive access devices (tracheotomy, gastrostomy or endotracheal tubes) and previous antimicrobial treatment (Dhodapkar & Henry, 1996; Zinner, 1999; Casanova-Roman, Rios, Sanchez-Porto, Gomar, & Casanova-Bellido, 2003). Clinically, Leuconostoc has been described to cause primary bacteraemia, pulmonary infections and empyema, peritonitis and infection of the peritoneal fluid, endocarditis, meningitis, osteomyelitis, ventriculitis, abscess and septicaemia. Most strains belong to the species mesenteroides, the isolation of Ln. lactis being rare although most species except Ln. mesenteroides subsp. cremoris are able to grow at 37 C (Barreau & Wagener, 1990; Aguirre & Collins, 1993; Giacometti, Ranaldi, Siquini, & Scalise, 1993; Kikuchi et al., 1994; Facklam & Elliott, 1995; Dhodapkar & Henry, 1996; del Nozal-Nalda, Jimenez-Mejias, Suarez, & Viciana, 1997; Jimenez-Mejias, Becerril, Gomez-Cia, del Nozal, & Palomino-Nicas, 1997; Nelson, 1998; Rodriguez, Saavedra, Fernandez-Jurado, & Prados, 1999; Schabereiter-Gurtner et al., 2001; Golan, Poutsiaka, Tozzi, Hadley, & Snydman, 2001; Gillespie, Symons, & Mc Donald, 2002; Vagiakou-Voudris et al., 2002; Deye, Lewis, Patterson, & Jorgensen, 2003; Fauchais et al., 2003). The source of these infections remain unknown, most of them being hospitally acquired. Skin represents a possible portal in cases with co-isolation from the skin flora and access to the bloodstream through the gastrointestinal tract has been also suggested. In two cases, the organism was isolated from the infant formula or the enteral feeding, indicating that these are possible sources (Dhodapkar & Henry, 1996). It is worth noting that vancomycin treatment might offer Leuconostoc a selective advantage in an ecological niche devoid of bacteria that would have normally inhibited their 

3.2. Antibiotic resistance Resistance of Leuconostoc to vancomycin is a general intrinsic feature and is linked to the presence of a pentadepsipeptide with a C terminal d-lactate instead of a d-alanine in the peptidoglycan (Delcour, Ferain, Deghorain, Palumbo, & Hols, 1999). The vancomycin resistance d -Ala-d -Ala ligase gene from Ln. mesenteroides has been cloned (Park & Walsh, 1997). In contrast, few reports are available on other antibiotics (Swenson, Facklam, & Thornsberry, 1990; Kikuchi et al., 1994; Herrero, Mayo, Gonzales, & Suarez, 1996; Cappelli, Barros, Camello, Teixeira, & Merquior, 1999; Zarazaga et al., 1999; Zinner, 1999; Katla, Kruse, Johnsen, & Herikstad, 2000; Holt, Al-Sheikh, & Shin, 2001; Deye et al., 2003). The studies were either partial or the antimicrobial agents tested and susceptibility methods used (broth dilution, disk diffusion or Etest) differed, so few concordant results are available. Converging studies on antibiotic susceptibility of dairy or human clinical isolates of Leuconostoc strains showed that they are resistant to fosfomycin, ‘‘old’’ quinolones, and glycopeptides. They are susceptible or give intermediate sensitivity to macrolides and tetracyclines. Nevertheless, the use of commercial starter culture bacteria in the production of dairy products could represent a potential for spread of genes encoding resistance to antimicrobial agents; the utilization of antibiotics in animal husbandry, horticulture or industrial processes might contribute to the emergence of antibiotic resistance among human isolates (Patel, 1999; Luh et al., 2000; Teale, 2002); Leuconostoc is recognized as an opportunistic pathogen and antibiotic-resistant strains of Leuconostoc were isolated from infection sites (Kikuchi et al., 1994; Cappelli et al., 1999). 3.3. Health-related metabolic activities Bile salt hydrolysis is dependent on intestinal bacterial actions on body secretions. This reaction has a facilitating effect for bile salt excretion but can also be involved in various illnesses. Bile salt hydrolase activity is absent in Ln. mesenteroides strains (Tanaka, Doesburg, Iwasaki, & Mierau, 1999). Biogenic amines are organic bases, which have been implicated in food poisoning incidents usually from the consumption of different kind of foods, including cheese, meat and fish products, and red wine. They are mainly generated by microbial decarboxylation of amino acids. Biogenic amine production in Leuconostoc has never been reported (Gonzalez de Llano, Cuesta, & Rodriguez, 1998; Bover-Cid & Holzapfel, 1999) so that



Leuconostoc strains are first–grade components of starter cultures used in the dairy industry. Leuconostoc produces d-lactate from carbohydrate metabolism as do Lactobacillus delbrueckii (Carr et al., 2002). Human adults metabolise d -lactate slowly so that the recommended daily intake should not exceed 100mgkgÀ1 body weight (FAO, 1966). Neither d nor dl -lactate should be used in infant foods, except for therapeutic purposes. The normal consumption of dairy products could not lead to an excess of d -lactate.

4. Characterization and taxonomy

The reader may refer to the reviews of Thunell (1995), Stiles and Holzapfel (1997) and Bjorkroth and Holzapfel (2003), which give a comprehensive view regarding the history of the genus Leuconostoc and the current taxonomy of LAB present in foods, respectively. Leuconostoc strains are non-motile and non sporeforming cocci, usually present as pairs or short chains that belong to the low G+C branch of the Grampositive bacteria. They do not possess arginine dehydrolase and catalase, are heterofermentative thus producing CO2 from glucose metabolism beside d -lactate and ethanol or acetate. They are facultative anaerobic bacteria, which harbour mesophilic characteristics and grow at 10 C. 

4.1. Identification, characterisation and biodiversity Phenotypic characters have been used for a long time to isolate and characterise Leuconostoc and, sometimes,

to differentiate between species or sub-species (Table 2). As it is difficult to identify species or sub-species unequivocally using these classical methods, new molecular techniques permit a reliable and consistent identification, including that of Leuconostoc. They are used alone or combined to estimate the molecular diversity and (or) for identification of species or sub. species (Gurtler & Mayall, 2001). They were taken into consideration to claim recommendations regarding species definition (Stackebrandt et al., 2002). The molecular diversity of 221 dairy strains has been estimated by RAPD (C - ibik, Lepage, & Tailliez, 2000). Strains were classified as Ln. mesenteroides or Ln. citreum and these data were confirmed using 16S rDNA sequence and 16S rDNA fragment amplification, except for one strain. Strains formerly described as Ln. paramesenteroides using phenotypic characters are indeed variants of Ln. mesenteroides, which did not produce slime, rather than W. paramesenteroides strains. Thus, it will be interesting to test strains described as W. paramesenteroides in other collections. DNA-based methods are becoming widely used for differentiation of Leuconostoc (Lee, Park, & Kim, 2000; . Schonhuber, Le Bourhis, Tremblay, Amann, & Kulakauskas, 2001; Matte-Tailliez et al., 2001; Pe! rez, Cardell, & Zarate, 2002; Randazzo, Torriani, Akkermans, de Vos, & Vaughan, 2002; dal Bello et al., 2003; Ennahar et al., 2003; Jang, Kim, Lee, & Han, 2003; Reeson et al., 2003). Thus, they permitted to describe the microbial community in Stilton cheese, Leuconostoc colonies being homogenously distributed in all parts observed (Ercolini et al., 2003) and in Salers cheese, Leuconostoc being predominant during manufacturing

Table 2 Presumptive identification of Leuconostoc by phenotypical tests General characters Gram-positive Cocci (ovoid-shaped), non motile, non-sporeforming Facultative anaerobic, catalase negative Vancomycin resistant Production of gas from glucose No arginine hydrolysis Production of d-lactate from glucose Additional characters Growth at 8 C, no growth at 45 C No growth at pH 4.8 Growth with NaCl 7% No H2S formation Acid production from: glucose (all strains); arabinose, arbutin, cellulose, cellobiose, fructose, galactose, lactose, maltose, mannitol, mannose, melibiose, raffinose, ribose, salicin, sucrose, trehalose, and xylose (variable within the species or subspecies) 



Features of some species or subspecies No production of slime from sucrose by Ln. mesenteroides subsp. cremoris, Ln. lactis, Ln. argentinum No acid production from fructose by Ln. mesenteroides subsp. cremoris (and some Ln. argentinum strains), from maltose by Ln. mesenteroides subsp. cremoris (and some Ln. gelidum strains) No malate decarboxylation by Ln. fallax


and ripening (Duthoit et al., 2003). Separation of 16S ribosomal DNA by Temporal Temperature Gradient Gel Electrophoresis (TTGE) is of peculiar interest because it permits a rapid identification of most bacterial species present in dairy products, including Leuconostoc (Ogier, Son, Gruss, Tailliez, & DelacroixBuchet, 2002). The distinction between the subspecies mesenteroides, dextranicum and cremoris of Ln. mesenteroides was confirmed using proteins patterns or ribotyping (Villani, Moschetti, Blaiotta, & Coppola, 1997; Pe! rez et al., 2002) whereas it was questioned by C - ibik et al. (2000) who proposed that these subspecies might be biovars, using other molecular methods. RAPD permits the identification of strains of the new species Ln. fallax (Barrangou, Sung-Sik, Breidt, Fleming, & Klaenhammer, 2002) although the 16S rPCRRFLP analysis, which allows the identification of nine Leuconostoc species, did not include the species fallax, filcuneum, and fructosus (Jang et al., 2003).

4.2. Recent evolution and up to date taxonomy Only four Leuconostoc species are included in the Bergey’s Manual of Systematic Bacteriology (Garvie, 1986), the species Ln. mesenteroides comprising the three subspecies mesenteroides, dextranicum and cremoris (Table 3). The two main facts concerning Leuconostoc are the creation of the genus Weissella that comprises W. paramesenteroides (previously Ln. paramesenteroides) and some heterofermentative species of the genus Lactobacillus (Collins, Samelis, Metaxopoulos, & Wallbanks, 1993), and the assignment of Ln. oenos as a new genus, Oenococcus oeni (Dicks, Dellaglio, & Collins, 1995). Additionally, eleven new species have been described (Table 3): Ln. gelidum and Ln. carnosum

473

isolated from meat products, Ln. citreum and Ln. pseudomesenteroides from clinical isolates; Ln. fallax from sauerkraut, Ln. argentinum from Argentine raw milk, Ln. gasicomitatum associated with meat spoilage, Ln. kimchii and Ln. inhae from kimchi, Ln. ficulneum from a ripe fig and Ln. fructosum (previously Lb. fructosus). Strains found in the microflora of the wasp Vespula germanica present 90% homology with known Leuconostoc species and may constitute a new taxon (Reeson et al., 2003).

4.3. Comments about methods of comparison Although molecular methods are useful for taxonomy and phylogeny of strains, phenotypic characters remain realistic and play a predominant role in food science (Section 9). Interest for strains used as tools in human activities is often linked to at least one important property and understanding the relationship between genotype and phenotype is a great challenge (O’Donnell, Embley, & Goodfellow, 1993; Morris et al, 2002). Thus, Ln. fallax strains were characterized using DNA-based methods (Barrangou et al., 2002) but could also be distinguished from Ln. mesenteroides by the malolactic reaction, which is absent in the former but present in the latter. This underlines the interest and the facility of phenotypic discriminatory features that could be used routinely and prior (or accompanying) to molecular based techniques, e.g. arabinose, raffinose and fructose utilization (C - ibik & Chapot-Chartier, 2000), vancomycin resistance, CO2 and dextran production, or citrate utilization. In the same way, the ability of concentrated non-proliferating Leuconostoc cells to acidify milk varied from pH 6.5 to 4.1 and permitted

Table 3 Species included in the genus Leuconostoc Leuconostoc species

Previous nomenclature

References

Ln. mesenteroides subsp. cremoris subsp. dextranicum subsp. mesenteroides

Garvie (1986)

Ln. lactis Ln. pseudomesenteroides Ln. carnosum Ln. gelidum Ln. fallax Ln. citreum Ln. argentinum Ln. gasicomitatum Ln. kimchi Ln. ficulneum Ln. fructosum Ln. inhae

Garvie (1986) Farrow et al. (1989) Shaw and Harding (1989) Shaw and Harding (1989) Martinez-Murcia and Collins (1991) Takahashi, Okada, Uchimura, and Kozaki (1992) Dicks, Fantuzzi, Gonzalez, Du Toit, and Dellaglio (1993) Bjorkroth et al. (2000) Kim, Chun, and Han (2000) Antunes et al. (2002) Antunes et al. (2002) Kim, Lee, Jang, Kim, and Han (2003)

Ln. amelibiosum

Lactobacillus fructosus



to discriminate between the 107 strains tested for applications in dairy technology (Demirci & Hemme, 1995). Similarly, a recent study proposed a characterization of the LAB strains on the basis of the neutral compounds produced in the whey (Mauriello et al., 2001). Some tools that are not suitable for characterization of species could nevertheless be used to describe a reduced group of strains, e.g. plasmid profile and carbohydrate fermentation to distinguish between physiologically related dextran-producing strains (Holt et al., 2001) or citrate metabolism, carbohydrate fermentation pattern and peptidase activities to select dairy strains for technological applications (ServerBusson, Foucaud, & Leveau, 1999). Clinical Leuconostoc strains were identified as vancomycin resistant (VAN), and lacking Leu-aminopeptidase (LAP) and pyrrolidonylarylamidase (PYR) activities using a disk method (Facklam, Pigott, Franklin, & Elliot, 1995). However, strains of dairy origin utilized Leu-containing peptides for growth and showed LAP activity on leucylparanitroanilide derivatives (Foucaud, Hemme, & Desmazeaud, 2001; Herreros et al., 2003). This discrepancy may result from the absence of external hydrolysis activity or the absence of transport of the substrate and further experiments are required to conclude about peptidase specificity.

5. Growth of Leuconostoc

5.1. Cultivation, maintenance and preservation of cultures 5.1.1. Media for isolation and enumeration When processing samples of LAB, chilled 0.1% peptone water or 0.9% sodium chloride should be used for dilutions, and should not be refrozen prior to plating (Bellengier, Richard, & Foucaud, 1997a, b; Foucaud et al., 2001). Phosphate buffer diluent results in lower recovery rates. Cell chains should be mechanically disrupted before bacterial enumeration (Lange, Plihon, Taillandier, & Riba, 1995; Bellengier et al., 1997a, b). The cultivation of Leuconostoc may be accomplished using enrichment broths and selective or non-selective media, depending on a need to isolate either a particular genus from a mixture of microorganisms or to maintain isolates in culture (Bjorkroth & Holzapfel, 2003). Various media satisfying the general nutritional requirements (also called elective) of Leuconostoc normally allow high recovery rates, without completely inhibiting other groups. The most common are APT, Briggs, MRS, La and BHIYE. Because of their low selectivity, their application is limited to associations dominated by one type or group of organisms. Selective media, which contain one or more restrictive factors, which still allow growth of Leuconostoc but inhibit unwanted bacterial

groups in a mixed population, have been proposed. However, no completely effective medium is yet available. Most selective media based on the organism’s ability to metabolise citrate have proven to be unsatisfactory because many organisms in the same habitat (e.g. Lactobacillus, Lactococcus lactis subsp. lactis biovar diacetylactis) also metabolise citrate and not all Leuconostoc strains utilize citrate (Section 7.2). The aciduric nature or their preference for microaerophilic to anaerobic conditions was taken into consideration. Inhibitory factors such as potassium sorbate (MRSS pH 5.7), thallous acetate (MRST pH 6.5), sodium azide (MSE), antibiotics such as vancomycin or tetracycline have been used, alone or in combination (Benkerroum, Misbah, Sandine, & Elaraki, 1993; Mathot, Kihal, Pre! vost, & Divie" s, 1994). Usual medium for cultivation of pure strains of Leuconostoc is the classical MRS medium modified or not by omission of citrate and meat extract (C. Divie" s, personal communication). Media for physiological studies of sugar fermentation pattern, gas production, dextran formation, citrate degradation and others have been developed (Bjorkroth & Holzapfel, 2003). A chemically defined medium was also elaborated, which fulfils the nutritional requirements of a representative number of Leuconostoc strains and allows rapid and heavy growth comparable to that in complex medium (Foucaud, Fran@ois, & Richard, 1997). When co-cultures of Leuconostoc with Lactococcus are involved, MRS containing 30 mg mLÀ1 vancomycin remains the more simple media to detect Leuconostoc, Lactococcus being inhibited by lower concentrations (minimum inhibitory concentration o 2.5 mg mLÀ1; Mathot et al., 1994). M17 containing 5% glucose could also be used since Lactococcus gives good-size colonies after 24 h while Leuconostoc requires 48h (Liu, Asmundson, Holland, & Crow, 1997). In contrast, when 5% sucrose is added to M17, the formation of slime permits detection of Leuconostoc colonies before Lactococcus colonies (D. Hemme, unpublished). Moreover, this medium supplemented with sucrose allows more rapid detection of Leuconostoc than MRS supplemented with vancomycin or MSE medium, that also contains sucrose but delays growth, probably because of the presence of sodium azide (0.075%). b-galactosidase (b-gal) positive strains of Leuconostoc developing as blue colonies can be distinguished from Lactococcus strains that are b-gal negative on MRS agar plates spread with 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside (X-Gal; Mathot et al., 1994; Bellengier et al., 1997a, b). This is also convenient for discrimination between b-gal-positive and b-gal-negative Leuconostoc isolates. In the presence of vancomycin-resistant Lactobacillus strains, LUSM medium containing vancomycin, tetracyclin and tomato juice should be utilized (Benkerroum et al., 1993).


Strains of Leuconostoc grow at 30 C like mesophilic Lactococcus starters but are favoured by a low temperature (Section 5.3.2). Most strains grow well at 10 C and even at 4 C (Hemme, unpublished; Hamasaki et al., 2003). In contrast, some strains were described as thermoduric as they resist pasteurization, their presence on the equipment favouring contamination of the milk, which is further pasteurized (Martley & Crow, 1993). 



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5.1.2. Preservation of cultures For short-term maintenance of dairy Leuconostoc, stab cultures (MRS agar containing 1% lactose) may be stored at 4 C for 1–2 weeks. Viability may be retained in litmus milk supplemented with 5% yeast extract and 5% glucose or in MRS broth containing 1% lactose and 10% glycerol as protecting agents for 6 months to 1 year at –20 C or over a longer period at –80 C. The use of exponential phase cultures gives maximum viability upon freezing (Bellengier et al., 1997a, b; Bjorkroth & Holzapfel, 2003). Lyophilization of dairy Leuconostoc in milk supplemented with 4% lactose resulted in good long-term survival and retention of metabolic characteristics. 

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5.2. Industrial production As complex laboratory media are often too expensive, other media are used by the industry and their composition is often not available (proprietary procedures). Papa.ın-hydrolysed casein or milk or whey based medium or the same medium as these utilized for Lactococcus (1.4% yeast extract, 4% tryptone, 9% lactose) may be used in batch cultures under regulated pH conditions (often pH 6.5) and controlled parameters (C. Bouillanne, personal communication; Accolas & Auclair, 1967; Vedamuthu, 1994; Champagne & Gardner, 2002). The use of lactose instead of glucose avoids the development of lactose-negative microorganisms. However, low yields (max. 5 Â 109 cells mLÀ1) were obtained compared with those obtained with Lactococcus or Lactobacillus in the same conditions (Vedamuthu, 1994). The type of centrifuge used to concentrate the cells did not seem to affect the viable counts recovery, but a continuous freeze-drying process had a negative effect (À33%) compared to a batch laboratory process (Champagne & Gardner, 2002). The production of mixed cultures containing strains of Lc. lactis, Ln. mesenteroides subsp. cremoris and Lb. rhamnosus, on commercial starter media has been assayed to replace blending mixture; population ratios, acidifying activities and aroma production being influenced by the incubation temperature (Barrette, Champagne, Roy, & Rodrigue, 2000).

475

5.3. Involvement of Leuconostoc in microbial interaction Mesophilic LAB starters used in the dairy industry are mixtures of genera, species, strains and even different variants. Their composition is thus not always known, particularly in the case of natural starters. In addition, microbial interactions, either beneficial (cooperation) or deleterious (inhibition) may lead to uncontrollable changes in the composition of the starter (Juillard et al., 1998). Positive interactions can result in a better growth or a better metabolite production. They are either direct, involving physical contacts of individuals or indirect, due to the modification of substrates or of medium parameters. Negative interactions can be direct and involved phages or they might also be indirect and concerned toxic metabolites (e.g. bacteriocin), competition for substrate consumption, and catabolite accumulation. 5.3.1. Co-aggregation reactions Extracellular material isolated from Ln. mesenteroides subsp. dextranicum 663 was shown to physically interact with lactococcal cells causing co-aggregation (Gopal et al., 1996). This may facilitate intergeneric interactions and (or) co-culture during cheese manufacture. 5.3.2. Growth and metabolism of Leuconostoc in mixed strain cultures Leuconostoc strains grow associatively with acidproducing lactococci and their associative growth has been studied with respect to citrate metabolism and aroma formation. It was described as a synergistic functional relationship (Walsh & Cogan, 1973; Boquien, Corrieu, & Desmazeaud, 1988; Vedamuthu, 1994; Monnet, Schmitt, & Divie" s, 1994; Jordan & Cogan, 1995). Leuconostoc, metabolized citrate over a pH range from 6.3 to 4.5, and diacetyl and acetoin are only produced at acidic pH (Section 7.2). Thus, to initiate citrate metabolism and to produce aroma compounds, sufficient acid production by Lc. lactis is required to depress the pH in milk (Cogan & Jordan, 1994). Less attention has been devoted to the growth rate, acid production and final biomass of these bacteria, which also reflect the interaction occurring in mixedstrain cultures of Leuconostoc and Lactococcus. Leuconostoc growth was either unchanged or was inhibited during the exponential phase and was systematically inhibited in the stationary phase (Vedamuthu, 1994; Bellengier et al., 1997a). Inhibition was usually suppressed upon addition of peptides or amino acids, showing that the competition for nitrogenous nutrients is a general feature of mixed-strain culture of Leuconostoc and Lactococcus in milk (Bellengier et al., 1997a). It is worth noting that, in limiting or excessive nitrogenous nutrient concentrations, Ln. mesenteroides was always disadvantaged over Lc. lactis (Foucaud et al., 2001;



Gendrot, Foucaud-Scheunemann, Ferchichi, & Hemme, 2002). In contrast, the improved maximum growth of Ln. lactis CNRZ 1091 in mixed-strain cultures with Lc. lactis subsp. cremoris AM2 in milk illustrated microbial cooperation between the two species (Boquien et al., 1988). Various types of interaction may occur depending on the strains used in the associative growth in milk but also on the growth temperature. A balanced growth of species occurs during incubation between 21 C and 25 C. At temperature above 25 C, the ratio is skewed toward the metabolically more active Lc. lactis, which grows at a faster rate at higher temperature (between 25 C and 32 C) relative to the Leuconostoc (Vedamuthu, 1994; Liu et al., 1997; Baron, Roy, & Vuillemard, 2000; Barrette et al., 2000). Differences in the growth pattern of the various strains constituting a mixed-strain starter can lead to unavoidable and important changes in its composition in a relatively short cultivation period. Moreover, in composite commercial culture concentrates containing acid-producers and aroma producing Leuconostoc, the proportion of Leuconostoc varied from 5% to 10% of the culture. Thus, the use of Leuconostoc strain concentrates may avoid the imbalance of population and the rapid dominance of Lc. lactis over Leuconostoc (Vedamuthu, 1994; Bellengier et al., 1997a). Although acetaldehyde (100 mg LÀ1) might enhance the production of acetoin and diacetyl as well as the growth of Leuconostoc, normal concentrations of acetaldehyde produced by mesophilic cultures (3 to 8 m g LÀ1) would be unlikely to stimulate the growth of Leuconostoc in mixed starter cultures (Vedamuthu, 1994; Cogan & Jordan, 1994). NSLAB may also influence the behaviour of Leuconostoc in dairy technology. Devoyod and Poullain (1988) reported the stimulatory action of enterococci and yeasts on Leuconostoc growth and gas production through the production of amino acids and peptides from the degradation of caseins. 

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

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5.3.3. Metabolic end products and interference with microbial growth The mechanism by which Leuconostoc inhibits microbial growth of LAB as well as spoilage or pathogenic strains has been also attributed to degradation products of carbohydrate and citrate metabolism (Dessart & Steenson, 1995; Caplice & Fitzgerald, 1999). The antimicrobial effects of organic acids, including lactic and acetic acids, affect the membrane properties such as the membrane potential and the integrity of the cell (Mans de Marion, Raffellini, Fantuzzi, & Gonzalez, 1995; Davidson, 1997; Alakomi et al., 2000; Cabo, Braber, & Koenraad, 2002). The antibacterial properties of diacetyl or ethanol are also described, although their effects are slight in usual lactic fermentations. In the presence of oxygen,

Leuconostoc accumulates hydrogen peroxide that could be inhibitory to some microorganisms (Condon, 1987). 5.3.4. Bacteriophages of dairy Leuconostoc The absence of reports indicating problems in dairy manufacture associated with Leuconostoc phages may explain why studies on such phages are rare. Additionally, the slow growth of Leuconostoc strains in mixed starter cultures might also account for the difficulty to recognize those phages in fermentations in which acid production is normal. However, phages active against Leuconostoc were isolated from dairy products (whey samples, cheese, etc.). They were characterized as to host range, morphology, structural proteins, and genome fingerprint and belonged to the Siphoviridae family (Boizet, Mata, Mignot, Ritzenthaler, & Sozzi, 1992; Davey, Ward, & Brown, 1995; Dessart & Steenson, 1995; Ackermann, 2001). 5.3.5. Bacteriocins produced by dairy Leuconostoc There are few reports on antimicrobial action of Leuconostoc against pathogenic and spoilage microorganisms in which the inhibitory activity is attributed to bacteriocin-like substances (Stiles, 1994). Studies are either old or preliminary, limited to the isolation of bacteriocin producers, and the incomplete characterization of antibacterial substances (Devoyod & Poullain, 1988; Serot, Dousset, Zucca, & Torcatis, 1990; Martley & Crow, 1993; Malik, Naveen, & Mathur, 1995; Sarkar & Sharmistha, 1996; Fantuzzi, Raffellini, & Gonzalez, 1999; Rodriguez, Gonzalez, Gaya, Nun ˜ez, & Medina, 2000; Brashears et al., 2003; Heikkila. & Saris, 2003; Pepe, Blaiotta, Moschetti, Greco, & Villani, 2003). Bacteriocins produced by some Leuconostoc strains belong to the subclass IIa bacteriocins that are pediocinlike, small heat-stable non-modified peptides, and active against Listeria (Ennahar, Sashihara, Sonomoto, & Ishizaki, 2000). Mesentericin Y105 and B105 and mesenterocin 52A and 52B from dairy Leuconostoc strains have been extensively described and their biological activity, structure and properties have been analysed (Mathieu, Sudirman, Rekhif, Millie" re, & Lef e" bvre, 1993; Sudirman, Mathieu, Benoit, & Lef e" bvre, 1994; Krier, Revol-Junelles, & Germain, 1998; He! chard, Berjeaud, & Cenatiempo, 1999; Ennahar et al., 2000; Corbier, Krier, Mulliert, Vitoux, & Revol-Junelles, 2001; Morisset & Fre" re, 2002). The mesentericin Y105 and B105 structural genes, mesY and mesB , respectively were cloned (Fre! maux, He! chard, & Cenatiempo, 1995; He! chard et al., 1999). Additionally, leucocin H (Blom et al., 1999) and dextranicin 24 (Revol-Junelles & Lef e" bvre, 1996) have been partly depicted. Different mechanisms, including the membrane fatty acid content or a putative immunity protein, induce resistance phenomena to bacteriocins in Leuconostoc


477

(Dayem et al., 1996; Limonet, Revol-Junelles, & Millie" re, 2002a, b). Although bacteriocins produced by Leuconostoc of dairy origin (mesentericin Y105) are being studied for their possible use in food preservation, the addition of bacteriocin from Leuconostoc or that of bacteriocin producers to food has never been reported. Actually, for optimal effectiveness against foodborne pathogenic and spoilage bacteria, bacteriocins may be used as part of a general multihurdle food preservation system, which involves a set of antimicrobial factors (Ennahar et al., 2000; Cleveland, Montville, Nes, & Chikindas, 2001).

6. Survival of Leuconostoc to stress conditions

Little is known about the effect of stress on Leuconostoc cells (Cibik & Chapot-Chartier, 2000). They are able to survive for a long time in unfavourable environments as diverse as the sugar, oil or dairy industries. They remained viable for many years on the surface of wooden ‘‘gerle’’, glazed sandstone, iron or plastic moulds used and other wooden or glazed tools used in traditional cheese making (Fig. 1; Devoyod & Poullain, 1988). Hostile environmental conditions favour interaction phenomena through the formation of slime or glycocalix in the presence of saccharose and trace minerals, resulting in a biofilm, which protects cells against detrimental agents (Kim, Thomas, & ScottFogler, 2000; Section 2). Curd of ripened cheese probably permits the survival of Leuconostoc that is detected at high cellular levels (Devoyod & Poullain, 1988; Mor-Mur, Carretero, Pla, & Guamis, 1994). In potassium buffer, the ability of non-proliferating cells to lyse was lower for cells grown in lactose or galactose containing MRS than in glucose containing MRS and varied with the strain from 7% to 44% after 24h at 30 C and pH 6.5, remaining stable thereafter (Cibik & Hemme, 1997; Cibik & Chapot-Chartier, 2000). In cheese technology, the strain dependency was also evidenced and the low lysis observed corroborated that observed in buffer, pH above 6.5 in the presence of Ca2+ and Na+, underlying the importance of those factors (Hemme, Bienvenu, & Ogier, unpublished; Turner, 1988; Martley & Crow, 1993). Two major peptidoglycan hydrolases (PGH), a glycosidase and an N -acetylmuramyl-l -Ala amidase (or endopeptidase) were described. Additional PGHs were only detected in Ln. lactis with Ln. mesenteroides cell wall as a substrate, suggesting that Ln. lactis strains can be separated from other species by their PGH profile. An atypical PGH, called LnMur, has been described in Ln. citreum 22R and the gene cloned. It lacks the amino acid repeats implicated in the cell wall binding. The encoded protein of 209 amino acid residues included a 31 amino acids putative signal peptide. A 

Fig. 1. Adhesion of Leuconostoc on glazed sandstone moulds, examination by scanning electron microscopy. Reproduced from Devoyod and Poullain (1988) with authorization of Le Lait.

chimeric fusion protein between LnMur and Lc. lactis AcmA C-terminal domain is able to complement AcmA deficiency in Lc. lactis AcmA (C - ibik & Chapot-Chartier, 2000; C ibik, Tailliez, Langella, & Chapot-Chartier, 2001). Homeostasis of the internal pH is essential for growth and survival of all biological cells, including Leuconostoc but bacterial growth is a self-limiting process through the acidification of the external medium and acid accumulation (Cogan & Jordan, 1994; Konings, 2002; Section 7). As pHout decreased, the pHin of Ln. mesenteroides decreased in contrast to that Lc. lactis. Growth ceased when internal pH values of 5.4 to 5.7 were reached, independent of the composition of the medium. In contrast, the limiting external cellular pH was greatly influenced by the growth medium, i.e. nature and concentration of organic acids. The response of Ln. mesenteroides cells to heat shock consisted in the overexpression of stress proteins (Salotra et al., 1995), the N-terminal sequence of which is homologous to the highly conserved chaperone proteins DnaK and GroEL of Escherichia coli . Ctsr homologues were identified in Oenococcus oeni and other Gram-positive bacteria, suggesting that heatshock regulation by Ctsr might also exist in Leuconostoc (Derre, Rapoport, & Msadek, 1999). Preservation technologies, such as high pressure technology, which combine efficient germ reduction with a maximal retention of the chemical and physicochemical product properties, are currently under investigation. High pressure homogenisation treatments at 25 C for 15 min and pressure ranging between 100 and 300 MPa showed that Gram-positive bacteria, including Ln. mesenteroides were more resistant than Gramnegative bacteria, the structure of the peptidoglycan probably contributing to the resistance whereas both 



groups overlapped at high hydrostatic pressure (Wuytack, Diels, & Michiels, 2002). The destruction of Leuconostoc depended both of the pressure level and the pressure treatment time, the pressure resistance of Ln. mesenteroides being lower than that of Saccharomyces cerevisiae (Basak, Ramaswamy, & Piette, 2002). After 5 min at 25 C and a pressure of at 345 MPa the cell viability loss was over 6 log cycles in 1 h. In the presence of pediocin AcH, cell wall degradation was faster and more extensive (Kalchayanand, Frethem, Dunne, Sikes, & Ray, 2002).

obtained energy by fermentation, always producing lactic acid as all other LAB and CO2 and ethanol or acetate as reviewed by Cogan and Jordan (1994). A large variety of mono- and disaccharides support growth of Leuconostoc strains (Vaughan, David, & de Vos, 1996; Server-Busson et al., 1999; Holt et al., 2001; Section 4). The sugar fermentation pattern (e.g. arabinose, xylose, ribose, and fructose) of the strains constitutes a phenotypical criterion for species, subspecies and strain characterization and is of great interest to choose strain for potential application (Section 4).

7. Metabolism of Leuconostoc

7.1.1. Sugar transport Leuconostoc takes up carbohydrates by permeases that permit the entry of sugars into the cell without modification of the substrate (e.g. phosphorylation when the PEP-PTS system is involved). The lactose transport gene, lacS from Ln. lactis has been characterized, using functional complementation of lactose permease-deficient E. coli mutants (Vaughan, David, & de Vos, 1996). It encodes a 24 kDa hydrophobic protein and presents significant homology with the Gln and His transport protein genes of enteric bacteria (Cogan, 1995).



The general metabolism of Leuconostoc is presented Fig. 2. 7.1. Carbohydrate utilisation Leuconostoc does not possess functional cytochromes and lacks some enzymes of Krebs cycle. They only

Fig. 2. General metabolism of Leuconostoc. Major products formed are indicated in bold. Numbers refer to enzymes involved or steps: (1) dextransucrase; (2) mannitol-dehydrogenase; (3) b-galactosidase; (4) esterase; (5) NADH oxydase; (6) alcohol dehydrogenase; (7) phosphoketolase; (8) phosphotransacetylase, (9) a-acetolactate decarboxylase; (10) acetate kinase; (11) a-acetolactate synthase; (12) nonenzymatic formation; (13) diacetyl reductase; (14) oxaloacetate decarboxylase; (15) lactate dehydrogenase; (16) citrate lyase; (17) malate dehydrogenase; (18) formation of aspartate; (19) malolactic enzyme; (20) ATPase.

7.1.2. Sugar catabolism The heterofermentative utilization of glucose follows the phosphoketolase (or pentose-P) pathway and leads to CO2, a C2 compound (ethanol or acetate with an oxidative mechanism) and pyruvate. Pyruvate is further reduced by a d-lactate dehydrogenase in d-lactate that is partly used for the peptidoglycan synthesis, thus reducing the quantity to be rejected by the cell and avoiding d-Ala synthesis (Section 3.2). The d-lactate dehydrogenase gene, d-ldh from Ln. mesenteroides has been cloned (Phalip, Dartois, Schmitt, & Divie" s, 1994). The final reaction might be affected by exogenous acetaldehyde, which is taken from the medium when present and utilized as an electron acceptor (Section 9.1.2). The proportion of the metabolites depends of the conversion of the acetyl-P formed from xylulose-5-P. The acetate kinase directs the flux to the formation of acetate with production of ATP, the additional energy being used for growth, whereas the phosphotransacetylase (PTA) produces acetyl-CoA that is used for biosynthesis or for reoxydation of NADH with production of ethanol by alcohol dehydrogenase. The pta gene has been cloned and appears not to be associated with acetate kinase gene, both genes being as a monocistronic transcript (Bourel et al., 2001). Leuconostoc ferments fructose to fructose-6-P, which then enters the pentose-P pathway and is converted to mannitol without the co-formation of sorbitol (von Weymarn, Hujanen, & Leisola, 2002). The oxidation of NADH to NAD+ is achieved by reducing fructose to


mannitol by an NADH-dependent mannitol dehydrogenase. This affects the cellular redox balance and favours the production of acetate instead of ethanol so that an ATP is gained. The enzyme is a tetramer of 4 identical sub-unit of about 38kDa (Aarnikunnas, Ronnholm, & Palva, 2002; Hahn, Kaup, Bringer-Meyer, & Sahm, 2003). The mannitol dehydrogenase gene (mdh) was cloned (Aarnikunnas et al., 2002; Hahn et al., 2003). The sequences of the gene and of the enzyme are distinct from those of other bacteria, hybridization occurred only weakly with some heterofermentative Lactobacillus strains. The question remained to know if this peculiarity is related to the use of fructose as an electron acceptor. The fermentation balance and pathway regulation have been explored (Grobben et al., 2001). Galactose and mannose are probably used via the Leloir pathway (Cogan & Jordan, 1994). The galactose genes are chromosomally encoded and the galK gene from Ln. lactis has been cloned (Grossiord, Vaughan, Luesink, & de Vos, 1998). Sequence analysis of the DNA flanking galK revealed an open reading frame (ORF) upstream with homology to a a-galactosidase gene and a downstream ORF with homology to galT from other LAB. The utilization of d-tagatose has been recently studied (Bertelsen, Andersen, & Tvede, 2001). Pentoses are converted to xylulose-5-P that is further catabolized in glyceraldehyde-3-P and acetyl-P. Neither ethanol nor acetaldehyde is produced and 1 mole of ATP and acetate are formed from acetyl-P (Cogan & Jordan, 1994). Lactose is cleaved to galactose and glucose by a b-gal encoded by two overlapping genes (lacL and lacM ), which are highly homologous (99%) to those of Lb. casei (Cogan, 1995). The two monosaccharides are then used via the Leloir and pentose-P pathway, respectively. Most strains grow in the presence of a low lactose concentration (20 g LÀ1), whereas few required a high concentration, equivalent to that in milk (50 g LÀ1) or more. In this case, the induction of the b-gal may limit growth (Hemme, unpublished). Raffinose utilization by Leuconostoc is a variable character and the enzymes involved (a-galactosidase and b-fructosidase) have been studied in term of fermentation balance and induction (Milli e" re, Mathot, Schmitt, & Divie" s, 1989; Pre! vost, Phalip, Huang, & Divie" s, 1993; Server-Busson et al., 1999; Carr et al., 2002). During sucrose-grown cultures, a large fraction of sucrose is converted outside the cell into dextran (Section 9) and fructose and does not support growth. The other fraction enters the cell, where it is phosphorylated by a sucrose phosphorylase and converted to glucose-6-P. The sucrose phosphorylase gene from Ln. mesenteroides has been cloned (Kitao & Nakano, 1992; Kawasaki, Nakamura, Ohmori, & Sakai, 1996). Some polysaccharides (e.g. cellulose) are catabolized (Carr et al., 2002) and dextrans are susceptible to

479

degradation (Eggleston & Legendre, 2003). Although the immunological detection of CcpA-like protein and the partial characterization of the gene has been reported in Ln. lactis (Luesink, Kuipers, & de Vos, 1998), few are known relative to the excretion of metabolic end products and to the regulation of sugar catabolism. 7.2. Organic acid metabolism Citrate and malate are the two major organic acids metabolized by Leuconostoc. Metabolism of the former is important for aroma formation and gas production in fermented dairy products whereas the latter is unimportant because milk lacks malate (Cogan & Jordan, 1994; McSweeney & Sousa, 2000; Konings, 2002). Citrate present in the milk (8 mmol LÀ1) and further in the cheese curd is not utilized as a sole energy source in Leuconostoc but is degraded in the presence of a fermentable sugar. As Leuconostoc grows around neutral pH, the dianionic form (Hcit2À) of citrate is taken up in symport with a proton. The Arg 425 of the permease is implicated in the transport process of citrate and S-enantiomers of other substrates with a 2-hydroxycarboxylate motif (Bandell & Lolkema, 2000; Bourel et al., 2001). Internally, Hcit2À is further degraded in acetate and oxaloacetate, which is decarboxylated to pyruvate, thus increasing the pyruvate pool. The addition of citrate to glucose both increases the specific growth rate and molar growth yield of Ln. mesenteroides. Under these conditions, lactate is produced both from citrate and glucose metabolism and exchange between citrate and lactate favoured citrate metabolism. Metabolic energy conservation and pH homeostasis by citrate metabolism in Ln. mesenteroides has been reviewed by Konings (2002). Utilization of citrate leads to the formation of diacetyl, which is an important component of the flavour of dairy products, and other compounds such as acetate, acetoin and 2,3-butanediol (McSweeney & Sousa, 2000). The part of the putative reactions involved in the formation of diacetyl remains unclear. Diacetyl could be produced via the diacetyl synthase although this enzyme has not been clearly detected in all LAB tested. Another way to obtain diacetyl is the oxydative decarboxylation of a-acetolactate formed from pyruvate that could occur spontaneously or may be proteinmediated. Recently, the acetolactate decarboxylase from Ln. lactis has been purified and sequenced (O’Sullivan, Condon, Cogan, & Sheehan, 2001). The co-metabolism of citrate plus xylose in Ln. mesenteroides results in a growth stimulation, an increase in d-lactate and acetate production and repression of ethanol formation (Schmitt et al., 1997). This correlates with the formation of acetyl-P from pentose (Section 7).



Genetic aspects of citrate transport and metabolism have been progressively studied. The gene encoding the citrate permease is localized on a 23 kb plasmid in Ln. mesenteroides, whereas it is chromosomal in Ln. lactis. Genes involved in the degradation of citrate are chromosomal and arranged in a clyR-mae-citDEF locus in Ln. mesenteroides (Bourel et al., 2001). The citrate permease (citP ) genes from Ln. lactis (Vaughan et al., 1995) or Ln. mesenteroides (Bourel et al., 2001), the citrate utilization gene cluster (cit CDEFG ) from Ln. mesenteroides or (cit MCDEFGRP ) from Ln. paramesenteroides (Martin, Corrales, de Mendoza, Lopez, & Magni, 1999; Bourel et al., 2001) and the gene of a putative regulatory protein, clyR (Bourel et al., 2001) were cloned. The diacetyl reductase has been purified and the gene cloned and sequenced (Rattray, MylingPetersen, Larsen, & Nilsson, 2003). Most LAB, including Leuconostoc but not Ln. fallax, carry out malo-lactic fermentation with the formation of l -lactate and CO2 from malate by malo-lactic enzyme (Cogan & Jordan, 1994; Barrangou et al., 2002; Konings, 2002). Recently, two open reading frames encoding for an NAD-dependent malic enzyme were found in the upstream region of the citrate utilization locus in Ln. mesenteroides (Bourel et al., 2001). 7.3. Nitrogenous compounds metabolism 7.3.1. Amino acid requirements Leuconostoc strains are fastidious organisms that require amino acids and vitamins in addition to a fermentable carbohydrate. Nutritional requirements for amino acids were determined by assessing growth in a chemically defined medium lacking one of the components. Although this varied considerably between species and strains, the branch-chained amino acids Leu, Ile and Val, as well as Gln were found to be essential (Cogan & Jordan, 1994; Bellengier et al., 1997b; Foucaud et al., 2001). Ser stimulated the growth of few strains, while Ala was not required by any. Branched chain amino acid biosynthesis genes shared high similarity with the genes of the BCCA operon of Lactococcus and have been cloned from Ln. mesenteroides subsp. cremoris. Additionally, a catabolic aacetolactate synthase gene is also present but is located outside the putative BCCA operon (Cavin, Dartois, Labarre, & Divie" s, 1999). Ln. mesenteroides can synthesise Asp from oxaloacetate via transamination, which can further be converted to Asn and participates in the biosynthesis of pyrimidines and purines (Konings, 2002). The ability of Leuconostoc to utilize amino acids independently or as a mixture in the presence or not of an added amino acceptor varied with the strains and the species, suggesting that Leuconostoc offer either a great potential for flavour generation for amino acid or would impact

slightly on both physiology and cheese ripening (Crow, Liu, & Holland, 1998; Tavaria, Dahl, Carballo, & Malcata, 2002; Liu, Holland, & Crow, 2003a). 7.3.2. The proteolytic system Leuconostoc grows poorly in milk since no strains exceed 5 Â 108 cfumLÀ1 (Cogan & Jordan, 1994; Demirci & Hemme, 1994; Bellengier et al., 1997b). Growth of Leuconostoc can be stimulated up to 109 cfu mLÀ1 when the non-protein nitrogen content (NPN) of milk is artificially increased by the addition of amino acids or peptides (e.g. amino acid mixture, yeast extract, etc). This indicates that they lack adequate proteolytic activities that might provide them with assimilable NPN (Vedamuthu, 1994; Bellengier et al., 1997a, b; Server-Busson et al., 1999). However, preculturing milk with proteolytic strains of Lc. lactis is not recommended for promoting the subsequent growth of Leuconostoc in cheese manufacturing because the NPN accumulated in precultured milk has been shown to be generally insufficient to stimulate high Leuconostoc growth (Foucaud, Furlan, Bellengier, Juillard, & Richard, 1998). 7.3.2.1. Protein utilization. In contrast with lactococci, little attention has been devoted to the proteolytic system of Leuconostoc. Though few strains displayed some caseinolytic activities, this did not contribute much to growth (Vafopoulou-Mastrojiannaki & LitopoulouTzanetaki, 1996; Bellengier et al., 1997b; VafopoulouMastrojiannaki, 1999; Herreros et al., 2003). The hydrolysis of caseins may result from the activity of intracellular housekeeping proteases detected in intracellular extracts (Vafopoulou-Mastrojiannaki, 1999) or released into the external medium upon cell lysis (Ezzat, El Soda, El Shafei, & Olson, 1993), which probably have restricted practical importance (if any indeed exist). 7.3.2.2. Peptide utilisation and transport. Ln. mesenteroides utilized a large set of di- and tripeptides and oligopeptides up to seven amino acids residues for growth (Foucaud et al., 2001), or in non-proliferating state (Hemme, Giraudon & Vassal, 1996; Lepeuple & Hemme, unpublished), the ability varying with the strains. No extracellular breakdown of peptides occurs during peptide transport as peptides are transported in the peptide form and peptidases are located intracellularly (Foucaud et al., 2001). Separate transport systems were shown to mediate the uptake of amino acids and their dipeptides in Ln. mesenteroides (Foucaud et al., 2001). Recently, an oligopeptide transport system specific for peptides containing at least four amino acid residues was characterized in Ln. mesenteroides. It was enhanced in the presence of Mg2+ or Ca2+ ions, and driven by an


energy-rich phosphorylated intermediate (GermainAlpettaz & Foucaud-Scheunemann, 2002). Although there were large differences between strains, the overall peptidase profiles of most Leuconostoc strains are comparable to those of Lc. lactis (Vafopoulou-Mastrojiannaki & Litopoulou-Tzanetaki, 1996; Server-Busson et al., 1999; Foucaud et al., 2001; Herreros et al., 2003). However, in cell-free extracts of Leuconostoc, carboxypeptidase activity was either significant or low or absent (Vafopoulou-Mastrojiannaki & Litopoulou-Tzanetaki, 1996; Macedo, Vieira, Po@as, & Malcata, 2000; Herreros et al., 2003), while such activity was definitively not observed using whole cells (Foucaud et al., 2001), suggesting the absence of a carboxypeptidase or that of appropriate transport system or the specificity of the existing peptidase. Aminopeptidase and dipeptidylaminopeptidase activities have seldom been characterized (Foucaud et al., 2001). 7.3.2.3. Amino acid transport and utilization. The transport of branched-chain amino acids in cytoplasmic membranes of Ln. mesenteroides has been characterized as a proton-motive-force driven system (Winters, Poolman, Hemme, & Konings, 1991). Recently, multiple amino acid transport systems were characterized using whole cells. Some of these systems were shared by several amino acids and their kinetic characteristics could be an additional tool to estimate the biodiversity of Leuconostoc (Gendrot, Ferchichi, Winters, & Hemme, 2000; Gendrot et al., 2002) although their activity does not limit growth. 7.4. Metabolism in the presence of oxygen In aerobic conditions, oxygen consumption is linked to an increase of growth yield and specific growth rate (Cogan & Jordan, 1994; Plihon, Taillandier, & Strehaiano, 1995, 1996). Oxygen is used as an alternative electron acceptor and is reduced to hydrogen peroxide and water, and acetate is largely substituted for ethanol (Condon, 1987; Nuraida, Grogolava, Owens, & Campbell-Platt, 1992; Cogan & Jordan, 1994). This is due to increased synthesis of NADH oxidase, which effectively regenerates reducing equivalents for continued fermentation and allows the acetyl-P to be used for ATP and acetate formation rather than reduction to ethanol as in co-metabolism of sugar and citrate (Section 7). The NADH oxidase from Ln. mesenteroides was described as a dimer or a tetramer with a subunit molecular mass of 53 or 55 kDa, respectively, depending probably of the strain and FAD as a cofactor (Koike, Kobayashi, Ito,& Saitoh, 1985; Sakamoto, Uchimura, & Komagata, 1996). Immobilized Ln. mesenteroides cells possessing high NADH oxidase activity have been successfully used for the regeneration of the coenzyme (Ergan, Thomas, & Chang, 1984).

481

The high intracellular levels of Mn2+ (ca. 6– 10mmolLÀ1) might provide Leuconostoc as some other LAB, with an important defence mechanism against endogenous OÀ 2 and other reactive oxygen species. This could be related to their original habitat where high Mn2+ concentrations are present and explained the absence of superoxydismutase or manganicatalase (Boyaval, 1989; Horsburgh, Wharton, Karavolos, & Foster, 2002). 7.5. Other metabolic considerations The esterase activities have been detected with enzymatic test strips (Biome! rieux, France) using bnaphtyl substrates (Millie" re et al, 1989; Hemme & Bellengier, unpublished). Leuconostoc showed a progressively lower hydrolysis rates as ester chain length increased from C4 to C12 substrates with C14 and C16 not being cleaved at all, and C18 being the best substrate. The hydrolysis of tributyrin, triolein, tributyrin or butter was not detected on Elliker-rhodamine b agar plates (Meyers, Cuppett, & Hutkins, 1996) whereas the hydrolysis of tributyrin was recently described in Ln. mesenteroides using naphthyl derivatives and postelectrophoretic detection (Katz, Medina, Gonzalez, & Oliver, 2002). Leuconostoc lactis synthesized mainly ethyl and butyl esters from tributyrin and ethanol during incubation in a broth via a transferase reaction (Liu, Holland, & Crow, 2003b). S-methylthioacetate was the only thioester formed by Leuconostoc cells incubated with methanethiol alone or in conjunction with various short-chain fatty acids (Lamberet, Auberger, & Berge" re, 1997). A d-2-hydroxycaproate dehydrogenase with broad substrate specificity, which converts 2-ketocarboxylic acids (including d-lactate i.e. 2-hydroxypropionate) in 2-hydroxycarboxylic acids while reducing NAD, has been found in Leuconostoc and in Lactobacillus strains . (Hummel, Schutte, & Kula, 1985). The question remains to know if this enzyme differs from the d-lactate dehydrogenase and could be involved in different parts of the metabolism.

8. Genetics of Leuconostoc

The genus Leuconostoc has not been subjected to extensive genetic research yet. Most genetic interest in Leuconostoc has focused on the instability of phenotypes essential in milk fermentations and on the transfer of genetic material, especially plasmid DNA, into Leuconostoc strains to improve their fermentation capacities as reviewed by Dessart and Steenson (1995).



8.1. Plasmid biology Leuconostoc harbours one or more native plasmids of various sizes. Lactose utilization, citrate permease activity (Dessart & Steenson, 1995), bacteriocin production (Fre! maux et al., 1995), diacetyl reductase (Rattray et al., 2003) are plasmid-linked but the majority of plasmids have no known function. The genetic organization and mode of replication of small cryptic plasmids have been recently described (Coffey, Harrington, Kearney, Daly, & Fitzgerald, 1994; Biet, Cenatiempo, & Fre! maux, 1999, 2002). The nucleotide sequence and structural organization of the plasmid pCI411 (2.9 kb) from Ln. lactis 533 (Coffey et al., 1994) and pFR18 (1.8kb) from Ln. mesenteroides FR52 (Biet et al., 1999) suggested that their replication is done by the rolling circle mechanism. The plasmid pTLX1 (2.7 kb) isolated from Ln. mesenteroides Y110 most likely replicates via a theta mechanism (Biet et al., 2002). All these plasmids may be useful for the construction of food grade vectors. 8.2. Cloning vectors and gene transfer There are no reports of cloning vectors developed specifically for Leuconostoc. They either represented existing antibiotic resistance plasmids or were developed from cryptic lactococcal plasmids (Platteeuw, Simons, & de Vos, 1994; Dessart & Steenson, 1995; Kleerebezem, Beerthuyzen, Vaughan, de Vos, & Kuipers, 1997; Bourel et al., 2001). Most of these vectors were efficiently introduced in Leuconostoc by conjugation or transformation, thus indicating the feasibility of genetic manipulations. The conjugative transfer of the transposon Tn919 from Enterococccus faecalis, nisin production, and lactose fermenting ability were performed and broadhost-range self-transmissible plasmids were used to establish and develop conjugal transfer protocols in Leuconostoc (Dessart & Steenson, 1995). Plasmidmediated DNA transformation by electroporation was optimized and used in molecular cloning experiments despite the frequencies of transfer remained sometimes low, suggesting the existence of a restriction modification system (Otts & Day, 1987; Dessart & Steenson, 1995, Bourel et al., 2001; Hemme & Vogel, unpublished). Natural (horizontal) gene transfer has been seldom examined in Leuconostoc. Insertion sequence elements were identified and analysed in Leuconostoc strains, suggesting the possibility of genetic exchange between these genera (Johansen & Kibenich, 1992; Vaughan & de Vos, 1995; Ward, Brown, & Davey, 1996). Amino acid sequences of bacteriocins produced by Leuconostoc are quite similar (e.g. mesentericin Y105 and leucocin A-UAL 187 from Ln. gellidum, Fleury et al., 1996) so

that Leuconostoc strains may constitute a good model for studying bacteriocin gene transfer and evolution. The vancomycin resistance genes in Leuconostoc are not the source of the high-level vancomycin resistanceassociated genes in enterococci as determinants differ (Delcour et al., 1999; Patel, 1999). The complete genome sequence of Lb. plantarum WCSF1 revealed that the gene cluster citR-mae-citCDEF is closely related to Ln. mesenteroides whereas the lactose permease LacS and b-gal are highly related to Ln. lactis (Kleerebezem et al., 2003). 8.3. Gene cloning and expression The first cloning studies concerned the chromosomal glucose-6-phosphodehydrogenase and phosphoglucose isomerase genes and a 16-kb restriction fragment of the lactose plasmid pNZ63 from Ln. lactis in E. coli . The Lc. lactis lacG gene and E. coli lacZ gene were expressed in W. paramesenteroides under the control of Lc. lactis, demonstrating the feasibility of recombinant DNA technology (Dessart & Steenson, 1995). The recA gene from Ln. mesenteroides has also been cloned (Duwat, Ehrlich, & Gruss, 1992). Since then, the cloned Leuconostoc genes reported so far increased and dealt with various aspects of technological interest. In the dairy field, this concerns bacteriocin production (Section 5.3.5), peptidoglycan hydrolase (Section 6), carbohydrate utilisation (Section 7.1), organic acid metabolism (Section 7.2), and nitrogenous compounds metabolism (Section 7.3.1). 8.4. Genomics The neotype strain Ln. mesenteroides ATCC 8293 that was isolated from fermenting olives and produced dextrans is currently sequenced by the Joint Genome Institute/Lactic acid Bacteria Genome Consortium (Walnut Creek, CA, USA; http://www.jgi.doe.gov/ JGI microbial/html/index.html). Basic facts and sequence annotation are in progress (draft sequence currently in pre-finishing phase): the genome size is 2.0 Mb in 116 contigs of 20 reads or greater with an average G+C content of 37.4%.

9. Roles of Leuconostoc in technology

Leuconostoc plays important roles in the technology of dairy products, in particular through the production of gas and aroma compounds. Despite often being present at high levels in the product with a stable population, all their roles are not clearly defined (Devoyod & Poullain, 1988; Alonso-Calleja et al., 2002) and other functions could be assumed in shelflife extension through the inhibition of undesirable



bacteria (Section 5) and in the new domain of functional foods. 9.1. Roles in dairy technology The role of Ln. mesenteroides subsp. cremoris in aroma production has been well described (Vedamuthu, 1994), but these of other Ln. mesenteroides is less recognized. A major reason is that for a long time Ln. mesenteroides subsp. cremoris, known for its aroma potential, was the unique adjunct available. In the same way, the literature reflects more the use of Ln. mesenteroides subsp. cremoris than that other species or subspecies mostly due to a lack of knowledge. Indeed, strains isolated from raw milk cheeses belong to the subspecies mesenteroides and dextranicum of Ln. mesenteroides and to Leuconostoc citreum but not to the subspecies cremoris of Ln. mesenteroides (Section 4.1; Devoyod & Poullain, 1988; Cogan et al., 1997; C - ibik et al., 2001). 9.1.1. Openness The demand for opening is the rule for Blue-veined cheeses and in particular Roquefort in which this allows the Penicillium roqueforti colonization. In soft ripened cheeses in which enough rigid curd cubes avoid collapse and thus permit mechanical opening, Leuconostoc creates an intracurd opening resulting from CO2 production. In pressed ripened Dutch cheeses such as Edam, Gouda and other brine salted cheese varieties, small and shiny openings are due to CO2 produced by Leuconostoc present in the starter and not to mechanical openness related to technological steps, i.e. pressing under the whey to avoid air entrapment. Strains are here selected for their moderate production of CO2 to avoid excessive openness (Vedamuthu, 1994; Martley & Crow, 1996). In Roquefort cheese production, Leuconostoc represents about 5% to 10% of the Lactococcus concentration, i.e. 5 Â 107 cells mLÀ1 of milk, the minimal concentration being 106 cells mLÀ1. Optimal results were obtained with selected strains of the subspecies mesenteroides of Ln. mesenteroides used as concentrated cells suspensions (Devoyod & Poullain, 1988; Pradel, personal communication). In a few cases, the population of Leuconostoc originally present in milk and coming from the environment creates the desired openness (Reiter et al., 1967; Devoyod & Poullain, 1988; Martley & Crow, 1993, 1996; Hemme, unpublished). In contrast, the addition of Leuconostoc is required with pasteurized and raw milk of increasing microbial quality, thus containing a low bacterial flora. The maximum concentration of CO2 produced is 16 mmol kgÀ1 of cheese, corresponding to the utilization of the 8 mmol LÀ1 citrate present in milk, but the activity varies both with the species and the strains (Bellengier,

Foucaud, & Hemme, 1993; Bellengier, Hemme, & Foucaud, 1994). Strains degrading citrate must grow during the early cheese making stages (pressing) although classical conditions (30 C) are unfavourable to Leuconostoc (Section 5.3). They must survive to continue this metabolism during ripening in the absence of sugar. However, some strains, active in low pH curds could be inactive in washed-curd cheeses having a higher pH (Turner, 1988; Martley & Crow, 1993). Leuconostoc has been involved in early and late blowing of some cheeses (Devoyod & Poullain, 1988). In Norwegian cheese of Gouda type, Leuconostoc strains were present in 7 out of 9 cheeses with atypical eye formation (Narvhus, Hulbaekdal, & Abrahamsen, 1992). 

9.1.2. Aroma production The major compound related to the utilization of Leuconostoc in the dairy field is diacetyl, acetate and ethanol contributing also to aroma formation (Vedamuthu, 1994). The level of diacetyl that is able to give the desired aroma is low (1.5 to 5 ppm) due to its low flavour threshold. Sufficient number of cells and peculiar physico-chemical conditions are required for optimal citrate utilization and aroma production (Sections 5.3 and 7.2). When allowed by legislation, addition of citrate to the milk (i.e. 0.15% in the USA) or in the wash water (i.e. when lactose removal technology is used, replacing part of the whey with water) permits a more intense aroma production. Leuconostoc may further transform diacetyl to acetoin and 2,3-butanediol, which do not give aroma. This unfavourable transformation could be lowered when products such as fermented milks are cooled after the aroma production. The storage of ripened cheeses is generally done at higher temperature (10–13 C), above those that are required to stop the process. The incorporation of oxygen in products such as fermented milks favours diacetyl production (Section 7). Excess acetaldehyde, which may be produced by starter cultures in butter and fermented milks leads to the development of a ‘‘green’’ flavour defect (Section 5). Under refrigeration, Leuconostoc is able to reduce acetaldehyde to ethanol, the activity being maximal for Ln. mesenteroides subsp. cremoris (Vedamuthu, 1994; Cogan & Jordan, 1994). In cheese, low pH, increased level of salt and low water activity reduce the acetaldehyde metabolism of Ln. mesenteroides subsp. cremoris (Liu et al., 1997). 

9.1.3. Deliberate addition of Leuconostoc as NSLAB Adjuncts of Leuconostoc comparable with those of other NSLAB (e.g. Lactobacillus) have been tested in different cheeses where they are not normally present since a real interest exists, in particular for raw milk flora (Crow et al., 2002).



Ln. mesenteroides subsp. cremoris strains usually used for butter, cream or fresh cheeses are not recovered when used in other cheese and have only a minor effect, if any. Ln. mesenteroides subsp. mesenteroides or dextranicum strains are preferred and the cell numbers in the products need to be at least 107 cells mLÀ1 of milk. Most strains gave low or no multiplication in the milk and in the curd, and this is not favoured by the presence of Lactococcus (Section 5). Milk can also be matured with about 106 cells mLÀ1, for 15h at 13 C, to reach this level (Hemme & Boulanger, unpublished). In Gouda-type cheese manufactured in aseptic industrial conditions, the effect was not clear despite a decrease in citrate content was observed (Martley, personal communication). On a pilot scale, pressedcheeses containing 108 cells gÀ1 present a higher pH (5.3 vs. 5.1), a higher amino acids content and an accelerated ripening than the control (Hemme et al., 1996; Hemme, . Boulanger Munchner, & Ogier, unpublished). This higher pH results from a slight inhibition of l-lactate production by Lactococcus and could partly explain the more rapid ripening observed. Leuconostoc could thus be a good candidate to counteract acidifying starters. These results were confirmed by trials in aseptic conditions using the micro-model developed for pressed type washed-curd cheeses (Hynes, Ogier, & DelacroixBuchet, 2000). Aroma was modified when cells concentrations were greater than 5 Â 106 cells mLÀ1 of milk, whereas openness appeared only between 5 Â 106 and 5 Â 107 cells mLÀ1, depending on the strain and relying possibly on its autolytic properties (Section 6). The texture of the product after two weeks was then equivalent to that in absence of Leuconostoc after 1 month so that the ripening process was accelerated (Hemme, Bienvenu, & Ogier, unpublished). Leuconostoc strain 5201 was also shown to inhibit NSLAB, which are involved in off-flavour production in Cheddar (Martley & Crow, 1993; Section 5). The inhibition by metabolic end products, combined with the reduced oxido-reduction potential that exists in cheese, has been described to inhibit Lb. plantarum (Ouvry, Wache! , Tourdot-Mare! chal, Divie" s, & Cachon, 2002). Ras cheese made in a pilot plant with addition of freeze- or heat shocked cells or cell free extracts of Leuconostoc strains isolated from the natural flora of French cheese, showed a decreasing bitterness (El Shafei, 1994). Manchego cheeses made from pasteurized milk using defined starters comprising Ln. mesenteroides obtained higher scores for flavour quality and intensity and overall impression that cheeses made with commercial starters (Poveda, Sousa, Cabezas, & Mc Sweeney, 2003). Very high numbers of cells in cheese trials with deliberately added cells (108 cells mLÀ1 of milk) give an 

excess of opening and concomitantly a typical silage offflavour (Hemme, unpublished). 9.2. Roles in functional foods A functional food comprises a component that serves to promote health or helps to prevent disease, and in general, the term is used to indicate a food that contains some health-promoting component(s) beyond traditional nutrients. This includes the addition of probiotics and the production of metabolites as well as activities that may contribute to the health benefit of food materials will also be considered hereafter (Hugenholtz et al., 2002; Ouwehand et al., 2003). 9.2.1. Leuconostoc as potential probiotics As some microorganisms currently proposed to the consumers, Leuconostoc does not colonize the intestinal tract, and their effect on the host through microbial actions is thus expected to be small except when ingested at high cell concentrations. Recently, studies of the feasibility of diarrhoea control in children by feeding fermented milk revealed that Indian Dahi containing 108 gÀ1 Lc. lactis and Ln. mesenteroides reduced the mean duration of diarrhoea by 0.3 day (Agarwal & Bhasin, 2002). 9.2.2. Production of polysaccharides Ln. mesenteroides produces exopolysaccharides (EPS), which are homopolysaccharide consisting of a-d -glucans such as dextrans mainly composed of a-1,6-linked residues with variable (strain specific) degrees of branching and alternans composed of a-1,3 and a-1,6 linkages. The biosynthesis process is external and requires sucrose. Specific glycosyltransferase and dextran or levan sucrase enzymes are involved in the biosynthesis process (de Vuyst & Degeest, 1999; Monchois, Willemot, & Monsan, 1999). Eight glucansucrase-encoding genes from Ln. mesenteroides were cloned (Bozonnet et al., 2002). The gene encoding the dextransucrase DsrD can be efficiently expressed and secreted in a heterologous host (i.e. Lc. lactis MG1363) and is able to drive dextran synthesis (Neubauer, Bauche, & Mollet, 2003). Recently, the gene coding for an inulosucrase in Ln. citreum was cloned, sequenced and expressed in E. coli. Bacterial inulosucrases, encoding for the production of inulin, have only been reported in Streptococcus mutans and Lb. reuteri , yet (Olivares-Illana, Lopez-Munguia, & Olvera, 2003). Industrial dextrans are currently used in the manufacture of gel filtration products and as blood volume extenders and blood flow improvers. In dairy technology, dextrans, as with other EPS, are used as food additives and act as texturizers by increasing viscosity and as stabilizers through strengthening the rigidity of the casein network by binding


hydration water and interacting with milk constituents. As a consequence, EPS decreases syneresis and improves product stability. They play a recognized role in the manufacturing of fermented milk, cultured cream, milk based dessert and flavoured milk (Pucci & Kunka, 1995; de Vuyst & Degeest, 1999; Duboc & Mollet, 2001; Cooke, Foster, Galema, Ledeboer, & Sanders, 2002). In addition to technological benefits, certain EPS are claimed to have beneficial physiological effects on the consumer. It is assumed that the increased viscosity of EPS containing foods may increase the residence time of ingested fermented milk in the gastrointestinal tract and therefore be beneficial to a transient colonization by probiotic bacteria (German et al., 1999). a-gluco-oligosaccharides produced by Ln. mesenteroides NRRL-B-18242, are highly resistant to attack by digestive enzymes. They have been proposed to have a potential prebiotic effect in human neonates as they stimulated the growth of beneficial bacteria of the intestinal flora (Djouzi et al., 1995). Such oligosaccharides were catabolized by bifidobacteria and lactobacilli but not by Salmonella or E. coli , pointing toward their effect on intestinal microflora modification (Chung & Day, 2002). 9.2.3. Production of mannitol Mannitol is a low-calorie sugar that could replace sucrose, lactose, glucose or fructose in food products. It is metabolized independently of insulin and is also applicable in diabetic food products. Ln. pseudomesenteroides and Ln. mesenteroides are known for their ability to produce mannitol in the fermentation of fructose (Section 7). Recently, high yields of mannitol have been reported, using a membrane cellrecycle bioreactor (von Weymarn, Kiviharju, & Leisola, 2002). 9.2.4. Hydrolysis of a-galactosides a-galactosides such as stachyose and raffinose that are commonly present in plants (including soya, maize and pulse derived food products) are not metabolized by humans nor animals owing to the lack of a-galactosidase in intestinal mucosa, hence causing flatulence. To overcome these drawbacks and to boost the consumption of otherwise highly nutritional food products, attempts have been made to eliminate a-galactosides using physical methods or a-galactosidase (Hugenholtz et al., 2002). Despite the ability of Leuconostoc to ferment a-galactosides (Boquien, Desmazeaud, & Corrieu, 1989; Pre! vost et al., 1993; Huang, Pre! vost, & Divie" s, 1994) and the proposal to use a-galactosidase from Ln. mesenteroides as a biotechnological approach to remove a-galactosides, attempts did not come out (Huang et al., 1994).

485

9.2.5. Production of vitamins Ln. mesenteroides strains producing significant amounts of menaquinones have been characterized and would be useful as starter cultures for dairy (and other) food fermentations or dietetary supplements to prevent vitamin K deficiency diseases (Morishita, Tamura, Makino, & Kudo, 1999). Recently, the production of folate (vitamin B9) has been reported in Ln. lactis and Ln. paramesenteroides (Sybesma, Starrenburg, Tijsseling, Hoefnagel & Hugenholtz, 2003).

10. Conclusion and prospects

10.1. Identification and ecology In the last few decades, new molecular tools, in particular DNA-based techniques, have contributed widely to clarify the phylogeny of Leuconostoc and to identify new species. Knowledge of microbial ecology in various niches and fermentations might also improve through the emergence of molecular identification and typing methods even though biochemical characters remain of major interest for technological applications. In addition, the undergoing genomic sequencing and analysis of LAB, including Leuconostoc will also allow a more comprehensive approach of both the potential and the relationships of Leuconostoc through genomics (Klaenhammer et al., 2002). 10.2. Health, safety and benefits Considering the wide distribution of Leuconostoc in the environment, the large numbers that are ingested daily in the vast range of fermented foods such as dairy products and the relatively few infections they cause, these bacteria have very little, if any, virulence for healthy human beings. No cases of infection have been directly linked to the consumption of fermented foods or to high occupational exposure to LAB. Even if the potential expansion in the area of functional foods would have to take safety aspects into detailed consideration (Feord, 2002), this history of human exposure and consumption has led to the reasonable conclusion that Leuconostoc is generally safe. The proper detection and characterization of clinical isolates of Leuconostoc as well as careful applications of infection control principles, judicious prophylaxis, and prompt effective therapy should maximize benefits for patients at risk of infection. Products containing Leuconostoc (as Lactobacillus) may also be a non-neglectible source of Mn2+ as cells contain millimolar concentrations of Mn2+and variants with increased concentration could be of potential interest (Boyaval, 1989).



Moreover, three aspects of Leuconostoc metabolism deserve considerations for the design of functional foods or the exploitation of metabolic properties in the context of industrial use. These are the ability of Leuconostoc (i) to hydrolyse a-galactosides such as raffinose from vegetable products, (ii) to synthesize acarbose analogues, which may improve the glycemic profile and insulin sensitivity in patients with type 2 diabetes (Yoon & Robyt, 2002) or (iii) to produce inulin-like polymers, which may serve as a prebiotic (Olivares-Illana, WacherRodarte, Le Borgne, & Lopez-Munguia, 2002). 10.3. Technological implications When considering their overall capabilities, Leuconostoc strains could be used to obtain a sufficient production of ethanol for ester production (Crow et al., 2002) or acetate for thioester, in cheeses in which methane thiol is present (Hemme, 1992), or for the conversion of acetaldehyde to ethanol and acetate (Liu et al., 1997). The fermentation of xylose or other pentoses will decrease the ethanol content of the product and enhance that of diacetyl, thus answering some demand for alcohol-free products. This metabolism, which occurs in vegetable fermentation, could also take place in mixed dairy-vegetable fermented food. The survival of most Leuconostoc strains in the cheese curd is rather high so that they can act as reactors for the production and (or) the consumption of metabolites during ripening. When considering their overall potential, Leuconostoc, as with other heterofermentative LAB, has a higher pyruvate dissipating potential than homofermentative LAB so that mutants could be obtained that reroute the pyruvate to the desired pathway. As with Lc. lactis subsp. lactis biovar diacetylactis, it could be possible to build up strains that would not transform the diacetyl produced. The butA mutant reported by Rattray et al. (2003) might be useful in improving the flavour stability of certain fermented dairy products, such as cultured buttermilk, sour cream, and ripened cream butter, where the characteristic butter flavour note is due to the presence of diacetyl. In the same way, if the production of d -lactate by Leuconostoc appears to limit its use, it could also be possible to obtain variants as with Lb. plantarum, that produce a low concentration of d -lactate, satisfying at least the synthesis of the peptidoglycan. Strains belonging to the subspecies mesenteroides of Ln. mesenteroides are more adapted to cheese manufacturing than Ln. mesenteroides subsp. cremoris strains commonly found in most commercial preparations. Efforts should be continued for an optimal selection based on technological potentials (aptitude to lyse, CO2 production, bacteriocin production, phage sensitivity, compatibilities with other starters, absence of biogenic

amines production, etc). In this context, manufacturers may increase the development of their proprietary starter cultures to fulfil the required quality linked to technological properties (e.g. flavour and texture) and to hygiene, as proposed for dairy fermentations (Poveda et al., 2003) and sauerkraut fermentation (Harris, Fleming, & Klaenhammer, 1992). Additionally, as adjunct cultures, Leuconostoc strains may also limit the growth and acid production of Lactococcus, thus avoiding negative impacts such as bitterness. They are also candidates as mixed-starter or pure cultures to mature the milk through the degradation of the NPN fraction and a slight acidification of the medium for subsequent fermentation and removal of the indigenous NPN fraction that may favour growth of potential contaminants. This concept would emerge if the use of milk treated by microfiltration expands in dairy plants.

Acknowledgements

The authors acknowledge the contribution of F. Martley (Fonterra Research Centre, Palmerston North, New Zealand), and J. Galle! (INRA, Jouy en Josas, France).

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Further reading Collins, Y. F., McSweeney, P. L. H., & Wilkinson, M. G. (2003). Lipolysis and free fatty acid catabolism in cheese: A review of current knowledge. International Dairy Journal , 13, 841–866. Kaup, B., Bringer-Meyer, S., & Sahm, H. (2003). Metabolic engineering of Escherichia coli : Construction of an efficient biocatalyst for d-mannitol formation in a whole-cell biotransformation. Applied Microbiology and Biotechnology , Papers in Press, published on line 28 October 2003, doi:10.1007/S00253-003-1470-9 . Menendez, S., Godinez, R., Hermida, M., Centeno, J. A., & Rodriguez-Otero, J. L. (2004). Characteristics of ‘‘Tetilla’’ pasteurized milk cheese manufactured with the addition of autochthonous cultures. Food Microbiology, 21, 97–104. Seifu, E., Buys, E. M., & Donkin, E. F. (2003). Effect of the lactoperoxidase system on the activity of mesophilic cheese starter cultures in goat milk. International Diary Journal , 13, 953–959. Sievers, M., Uermosi, C., Fehlmann, M., & Kriger, S. (2003). Cloning, sequence analysis and expression of the F1F0-ATPase beta-subunit from wine lactic acid bacteria. Systematic and Applied Microbiology, 26, 350–356.

Leuconostoc Review Denis Hemme

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