Lactococcus Piscium : a Psychotrophic Lactic Acid Bacterium with Bioprotective Or Spoilage Activity in Food - a Review

Journal of Applied Microbiology Achimer October 2016, Volume 121, Issue 4, Pages 907-918 http://dx.doi.org/10.1111/jam.13179 http://archimer.ifremer.fr http://archimer.ifremer.fr/doc/00334/44517/ © 2016 The Society for Applied Microbiology

Lactococcus piscium : a psychotrophic lactic acid bacterium with bioprotective or spoilage activity in food - a review

Saraoui Taous 1, 2, 3, Leroi Francoise 1, * , Björkroth Johanna 4, Pilet Marie France 2, 3

1 Ifremer, Laboratoire Ecosystèmes Microbiens et Molécules Marines pour les Biotechnologies (EM3 B),; Rue de l'Ile d'Yeu 44311 Nantes Cedex 03, France 2 LUNAM Université, Oniris; UMR1014 Secalim, Site de la Chantrerie; F-44307 Nantes, France 3 INRA; F-44307 Nantes ,France 4 University of Helsinki; Department of Food Hygiene and Environmental Health; Helsinki ,Finland

* Corresponding author : Françoise Leroi, tel.: +33240374172; fax: +33240374071 ; email address : [email protected]

Abstract :

The genus Lactococcus comprises twelve species, some known for decades and others more recently described. Lactococcus piscium, isolated in 1990 from rainbow trout, is a psychrotrophic lactic acid bacterium (LAB), probably disregarded because most of the strains are unable to grow at 30°C. During the last 10 years, this species has been isolated from a large variety of food: meat, seafood and vegetables, mostly packed under vacuum (VP) or modified atmosphere (MAP) and stored at chilled temperature. Recently, culture-independent techniques used for characterization of microbial ecosystems have highlighted the importance of L. piscium in food. Its role in food spoilage varies according to the strain and the food matrix. However, most studies have indicated that L. piscium spoils meat, whereas it does not degrade the sensory properties of seafood. L. piscium strains have a large antimicrobial spectrum, including Gram-positive and negative bacteria. In various seafood, some strains have a protective effect against spoilage and can extend the sensory shelf-life of the products. They can also inhibit the growth of Listeria monocytogenes, by a cell-to-cell contact-dependent. This article reviews the physiological and genomic characteristics of L. piscium and discusses its spoilage or protective activities in food.

Keywords : lactic acid bacteria, biopreservation, spoilage, meat, seafood, cold adaptation.

Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site. 2

Introduction

Lactic acid bacteria (LAB) constitute a heterogeneous group of Gram-positive bacteria, primarily non- sporulating, anaero-aerotolerant, and producing lactic acid as the principal end metabolite from carbohydrate fermentation. LAB can dominate the natural microbiota of many fermented foods where they play a key role in the development of the sensory properties (flavor and texture) and safety. In an appropriate environment, LAB can also colonize non-fermented products from plant or animal origin (Stiles and Holzapfel, 1997). Among LAB, the genus Lactococcus, and

Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site. known forthe ability to produce Bacilli lactis Pediococcus Although many bacterial species produce lactic acid, the LABgroup is restricted to fourteen genera, five of them constituting the core group ( Taxonomy This review deals with the characteristics of this species and its importance in food. spoilage microorganism depending on the strains and food matrix in concern. species is gaining the interest of scientists. 1990). However, during the last 10years its presence has been reported in various food and this isolated and characterized for the first time from diseased rainbow trout in 1990 (Williams etal., fujiensis, significantly distinct phylogenetic groups (bootstrap Phylogenetic analysis performed by theauthors has revealed that the genus Lactococcus hircilactis Lactococcus taiwanensis described: garvieae, Lactococcus raffinolactis and Lactococcus plantarum. basic physiological studies. Five species were initially described : group (lactic streptococci), according DNA-DNA 16S to hybridization, rRNA gene sequencing and al. (1985) to reclassify some species of the genus This article isprotected All rights bycopyright. reserved. Accepted Article importance for the food bio-transformation industry (Stiles, 1996). The species particularly (subsp. , Order L. garvieae Lactococcus chungangensis Lactococcus lactis, and lactis, cremoris, tractae Lactobacillales Streptococcus and L. formosensis and Lactococcus and laudensis (Chen et al., 2013), andFamily has been extensively studied, as some species are of major economic ). The genus The ). L -lactic acid from glucose. and hordniae (Cho et al., 2008),(Cho etal., formed the first group whereas Streptococcaceae L. piscium Lactococcus Lactococcus formosensis Lactobacillus, Lactococcus, Leuconostoc, Streptococcus (Meucci et al., 2015) ), as well as ≥ is described either as a bioprotective or a 98%) (Figure 1). The four subspecies of belongs to the phylum Lactococcus fujiensis (Samarzija et al. 2001). This genus is Lactococcus formerly included inthe N-Lancefield L. taiwanensis Recently, six new species have been L. lactis, L. piscium, Lactococcus (Chen et al., 2014),(Chen etal., . L. laudensis was proposed by Schleifer et

Lactococcus , L.hircilactis L. piscium L. piscium (Cai et al., 2011), Firmicutes , L. raffinolactis formed two was was , Class L. , L. L. , This article isprotected All rights bycopyright. reserved. L. chungangensis Biochemical, physiological, andgenetic aspects of decades by the dairy industry, andhas thus been extensively studied as amodel microorganism. the Qualified Presumption of Safety (QPS) approach generally recognized as safe (GRAS)by theUSF al., 2010), probiotics (Daniel et al., 2009) and protective cultures (Sarika et al., 2012). piscium on thirty lactococci genome revealed 3major clades: (i) species of environmental or animal origin ( reliable tools in species level identification of meat lactococci. In addition, the pangenome tree made EcoRI and genes 16S rRNAgene sequences of twenty-two LAB strain (2012) whoobtained two distinct phylogenetic groups of Lactococcus value of92% and 99% sequence similarity. This result confirmed those obtained by Rahkila etal. phylogeneticanalysis indicated that intestine microbiota. However, inmarine farmed fish, other piscium Its direct involvement in the disease has never been evidenced and, to the best of our knowledge, L. Habitat Some strains, mainly L. lactis Accepted(Boucher et al., 2003; Alomar etal., 2008 four species initially described in the genus: a review see Von Wright, 2012; Cavanagh et al., 2015). Over theyears, interest has grown in the other Article rpo , L.raffinolactis, L.chungangensis has never again been isolated or identified by culture-independent techniques in the fish L. piscium subsp. A and phe Cla I ribopatterns andphylogenetic sequence analyses of cremoris strains (Andreevskaya etal., 2015). , L. plantarum was described for the first time by Williams etal. (1990) in diseased rainbow trout. S of seventy-oneIn LAB. thesame study, they showed that numerical analyses of L. lactis , are widely applied in industrial processes as starter cultures (Kelly et and L. piscium grouped close related together. Interestingly, this this Interestingly, together. related close grouped L. piscium ; Matamoros et al., 2009a; Rahkila et al., 2012). and L. garvieae L. clustered with L.plantarum L.garvieae DA and considered by theEFSA tobe suitable for L. lactis L. lactis to safety (EFSA,2011). It hasbeen used for s and (ii) partial sequences of thehousekeeping , L. raffinolactis L. lactis L.lactis (ii) ); are widely described in the literature (for Lactococcus rpo using two different analysis (i) A and phe , L.plantarum species have been shown to subsp. lactis supported by a bootstrap S genes were strains and (iii) and L. lactis L. lactis

L. piscium L. piscium is is L. under MAP (70% O shrimp). sausage)seafood andfourproducts (salmonand communities associated with four meat products (ground veal and beef, diced bacon andpoultry rRNA gene pyrosequencing on160samples of fresh and spoiled foods to compare the bacterial other products, although strains have not always been isolated. Chaillou et al. (2015a) conducted 16S More recently, the use of culture independent techniques has revealed the presence of minimally processed vegetable salads (Delhalle et al., 2016; Pothakos et al., 2014a,b). turkey sausage (Kesmen et al., 2012), raw and cooked Belgiummeat, tartar steak andready-to-eat al., 2009a; Macé et al., 2012), and VP MAP pork (J skinned and boned broiler products (Vihavainen et al., 2007), raw salmon under MAP (Matamoros et et al., 2015; Jääskelänen et al., (MAP) and vacuum packed (VP) food (Table 1), in During the last 10 years, as a fish commensal andalso anopportunistic pathogen (Michel et al., 2007). hemorrhages (Eldar et al., 1996; Vendrell et al., 2006) Genomic characteristics of 2011), as well as in human feces by rRNA-Targeted Reverse-PCR (Kubota et al., 2010). multiplex PCR (Odamaki etal., 2011), and cheese, using 16SrRNA library sequencing (Carraro et al., The presence of L.piscium This article isprotected All rights bycopyright. reserved. be involved in epizoothics, such as salmon fillets (50% O Accepted(Morita et al., 2011), (Bolotin et al., 2001; Makarova etal., 2006), Article The complete genomes ofnumerous L. piscium was the dominant species in ground veal andground beef stored at 4 and 8°C 2 , 30% CO L. piscium 2 , 50% CO L. piscium has also been reported in dairy products such as raw milk, using anovel Lactococcus piscium Lactococcus 2 2016), MAP marinated broiler meat leg (Björkroth et al., 2005), MAP ). (Marché etal.,2014; Andreevskaya2015),al., et 2 ) and in the top twelve inCSS(Chaillou et al., 2015b). L. piscium L. garvieae has been isolated in avariety ofchilled, modified atmosphere was also in the top five species of the microbiota of MAP Lactococcus L. raffinolactis responsible for septicemias, opthalmias and cod fillet, cold-smoked-salmon (CSS)and cooked

cluding beefmeat (Sakala et al., 2002a; Ferrocino iang etal.,2010; Rahkila et al., 2012), fermented

and L. raffinolactis species, have been sequenced e.g. (Meslier etal.,2012), L.garvieae , which has been identified

L. fujiensis L. piscium L. lactis in pairs or in short chains. Ascanning electron microscopy picture is presented inFigure 2.Cai et al. motile cocci indiameter. from Cells 0.5to1 are sphericalµm or ovoid and appear individually, in This article isprotected All rights bycopyright. reserved. Physiological characteristics of miscellaneous categories (Marché et al., 2014). represent transporters, regulators and factors, and 16% fall under components of cell processes and CDS-encoded proteins are classified as prot plasmid of 20 AnnotatedKb. genomic nucleotide sequences are not accessible yet. A major part of its size is ~ 2.26 Mbwith 2239 CDSandGC%content is 39%.It contains one chromosome andone (vi) proteins of unknown function motility; (iv) metabolism, energy production and conversion; (v) signal transduction mechanism and cell and biogenesis envelope division, cell (iii) and repair; DNAreplication and structure ribosomal proteins involved in primary andsecondary metabolism andtransport; (ii) transcription, translation, LN774769-LN77477. The functions of deduced CDS-encoded proteins have been attributed to (i) through the European Nucleotide Archive (htt 55671 bp (66 CDS) and 53257 bp(64 CDS).Annotated genomic nucleotide sequences are accessible content is 38.79%. It contains one chromosome raw salmon (Marché et al., 2014). The CNCM I-4031 (also named strain of meat isolated from MAPbroiler fillet strips (Andreevskaya etal., 2015) and size variability upto600 kbp even within kbp. Genome-based analysis performed by Passerini et al. (2010) revealed that there are agenome Sequences (CDS). This indicates variability ingenome size between species of species have genomes ranging between1950 to2641kbp andcontained 1947 to 2476 coding DNA (http://www.ncbi.nlm.nih.gov) Accepted ArticleConcerning L.piscium L. piscium is a facultative anaerobic, Gram-positive, catalase- andoxidase-negative andnon- , two different strains have been sequenced: L. piscium andL.chungangensis Lactococcus piscium (Andreevskaya etal., 2015). The EU2241), a bioprotective strain in seafood isolated from MAP L. piscium MKFS47 genome size is~2.5 Mb andGC% L. lactis eins of unknown function, 24% as enzymes, 20% p://www.ebi.ac.uk/ena) under accession numbers with 2394138 bp (2289 CDS)and twoplasmids with subsp. lactis (https://www.patricbrc.org)

strains. strains. L. piscium L. piscium CNCML. piscium I-4031genome MKFS47, aspoiling Lactococcus up to 750 (Table 2). Those L. piscium able to hydrolyze aesculin but not arginine and they have nourease activities. Starch hydrolysis is CoA,NAD, polyprenylslipoate folate, and (Andreevskaya 2015). al., The et except phenylanine, of purine/pyrimidine and also several cofactors/vitamins, such asriboflavin, addition to these catabolic capacities, nucleic bases (adenine, guanine and uracil) and avitamin (riboflavin) (Saraoui et al.,2016). In cysteine, histidine and glycine and, in lesser quantities, isoleucine, lysine and leucine, aswell as the medium” (MSMA)containing glucose and other components showed that this strain catabolizes strains can use saccharose, trehalose, arbutin, fermented: glucose, fructose, lactose, galactose, gluconate, gentiobiose, mannose, maltose, melobiose, have been tested according tothe literature, authors have shown that the following carbohydrates are fermentative bacterium that canferment many carbonsources. Although only few et al., 2002b; Andreevskaya (Cai et al., 2011; Leroi et al., 2012; Meucci et al., 2015). higher. Leroi et al. (2012) later demonstrated that the NaCl This article isprotected All rights bycopyright. reserved. (2011) andSakala et al. (2002b) showed that L.piscium A metabolic profile of that obtained onglucose. piscium (saccharose, maltose, lactose, trehalose and cellobiose). These authors also demonstrated that degradation of monosaccharides (glucose, fructose, mannose, mannitol and xylose) anddisaccharides Andreevskaya etal. (2015) has shown that many Andreevskaya etal., 2015). Sequencing andannotation ofthe genome ofL.piscium al., 2013). The fermentation ofribose is variable and strain-dependent (Sakala et al., 2002b; mannoside, mannitol and amygdalin (Williams et al Accepted Articlewas 23g.l Biochemical analysis, metabolic profiling andthe MKFS47 cangrow onglycerol as aunique carbon source with a growth rate comparable to -1 . The optimal pH forgrowth was neutral and L -arabinose, N L. piscium D et al., 2015; Saraoui et al., 2016) revealed that -turanose, -acetylglucosamine, salicin,and strain CNCMI-4031 ina synthetic medium called “modified shrimp L. piscium D -xylose, melezitose, harbors genes for the biosynthesis ofall amino acids, catabolic pathways are predicted, including the ., 1990; Sakala et al., 2002b; Fall, 2011; Chen et genome analysis (Williams et al., 1990;Sakala wasnot able togrow at 30 g l L. piscium max α growth of -methyl- D could not grow atpHbelow to 4.8 -raffinose. In addition, some D -glucoside, -glucoside, L. piscium L.piscium L. piscium L. piscium CNCM I-4031 CNCM MKFS47 by α -methyl-D- -1 is a homo- NaClor strainsare strains strains L optimumgrowth temperature around 30°C (Cavan (Kubota et al., 2010). All the other Lactococcus Andreevskaya etal., 2015), it is noteworthy that absence of growth at 37°C (Williams et al., 1990; Sakala et al., 2002b; Leroi et al., 2012; fresh beef)VP anddevelopment wasweak andvariable at30°C (Sakala et al., 2002b). Despite the (Williams et al., 1990). Growth at 5°C wasobserved for all of the 20 strains tested (isolated from failed to grow at 30°C (Leroi et al., 2012) whereas L.piscium Growth at 30°C isweak and variable among thestrains. Another below 27-29°C, whichis not common among (Mat LAB grow at0°C withan optimumgrowth temperatur plates incubated at 8°C (Matamoros et al., 2009a). chilled VP or MAP meat and seafood. Several strains of temperatures have been recently tested more frequently, enumeration temperature commonly usedforLA Papadimitriou, 2011). Cold-adaptation of Lactococcus piscium al., 2015). metabolite is produced by the strains R-46592 and MKFS47 (Pothakos et al., 2014c; Andreevskaya et dependent. In fact, the strain CNCMI-4031 doesnot produce acetic acid (Fall, 2011) whereas this This article isprotected All rights bycopyright. reserved. slow and weak while H Accepted Articlepreservative agents such as organic acids, and high CO various steps in industrial processes, such as low the food industry. For example, these bacteria can survive different environmental stresses caused by The adaptation of LABtodifferent environmental conditions makes them ofgreat importance in L. piscium 2 S is not produced (Williams et al.,1990). Acetic acid production is strain- has long beendisregarded in food, probably because the

species aremesophilic microorganisms with an L. piscium L. piscium e at 24-26°C andamaximumgrowth temperature B enumeration has been 30°C. Since lower temperature, high salt concentration, presence of agh et al., 2015;al., Meucciaghet etal.,2015). L. piscium L. piscium 2 concentrations (Tsakalidou and amoros et al., 2009a; Leroi et al., 2012). L. piscium L. piscium has been isolated from human intestine type strain grew at 30°C butnot at 35°C is apsychrotrophic species, able to L. piscium has been isolated in various various in isolated been has were isolated on Elliker agar isolated from rawsalmon L. lactis, L. L. lactis, internal reactions between food components, reactions of the components with water and air and, odor, flavor andtexture.deterioration The fresof Role of Role preservation. especially against spoilage and pathogenic psychrotrophs, providing apromising perspective for food Cold-adaptation constitutes animportant advantage for bacterial competition in chilled food, and Marahiel, 1998). processes such as general stress, cellular growth, nutrient stress and the stationary phase (Graumann 2010). This can be explained by thefact that the Csp proteins play asignificant role inmany cellular responses and in fatty acid and energetic metabolism wasenhanced in cold conditions (Garnier et al., cold-adaptation (5°C) showed that the production of proteins involved ingeneral and oxidative stress Graumann andMarahiel, 1998). The comp up-regulated in response to cold-shock anddid not persist after the stress (Michel et al., 1997; In otherpsychrotrophic bacteria, such as Pseudomonas fragi and expression has been shown not to be regulated by cold-shock (Matamoros, 2008; Garnier et al., 2010). coding for the major cold-shock protein (CspE protein) waspresent in the L.piscium in the cold-shock response are constitutively produced. This is supported by the fact that the gene psychrotrophic LAB(Hamasaki et al., 2003). The speci were carried out at 5°C, after apre-culture at 26°C, contrary tomost similar (same growth rate and nolag phase). Additionally, nolag phasewas observed whencultures 4031 at its optimumgrowth temperature (26°C) and after a cold-shock (0or 5°C for1to 2h) were to cold temperatures. Garnier et al. (2010) showed that thegrowth kinetics of This article isprotected All rights bycopyright. reserved. garvieae, L. plantarum Accepted Article characteristic may help to differentiate L.piscium The quality offood can be determined by different sensory parameters such as appearance, The unusual temperature-growth profile among Lactococci suggests that Lactococcus piscium and L. raffinolactis L. in foodspoilage continued to grow at35°C arison of proteome profiles of hness occurs progressively during storage due to from other Lactococcus fic result suggested that the proteins involved Bacillus subtilis Lactococcus (Leroi etal.,2012). This L. piscium species. L. piscium L. piscium speciesother and , the Csp protein was at26°C andafter genome butits CNCM I- CNCM is adapted 8°C underMAP (50% CO strains into sterile or low contaminated food matrixes (Table 1). In sterile raw salmon fillets stored at spoilage effect of This led to the concept of the specific spoilage organisms (Dalgaard, 1995; Leroi et al., 2015). The established that in a food microbial ecosystem, only somemicroorganisms are involved in spoilage. its spoilage capability has to bedemonstrated by challenge tests. In fact, it has clearly been L. piscium et al., 2015a). became dominant at the time ofspoilage inmeat, and LAB and 2012). Two strains2012). Two of Fall etal.,2012;LeroiIn etal.,pork,2015). performed with different strains of L.piscium 2013). This low spoilage effect and characteristic off-odors are in accordance with other studies lightly spoiled by 44%. The weak odors associated were buttery and/or fatty fish–like (Macé et al., This article isprotected All rights bycopyright. reserved. - 22%CO operational taxonomic units drastically decreased during MAPandVP storage. LABand As anexample, in160 samples of various me caused by these technologies gradually reduces the number ofspecies present at the time ofspoilage. temperature, VP, MAP or addition of preservative ag strategies have already been adopted to prevent or delay thisdegradation, such as storage at chilled food production in sub-Saharan Africa (230 million tons) (Gustavsson et al., 2011). Asa result, some at the consumer level in industrialized countries (222 million tons) is almost ashigh asthe total net process leads to significant economic losses and is a major problem for the food industry. Foodwaste products become spoiled and unfit for human consumption and therefore have tobe discarded. This mainly, thegrowth and metabolic activity ofuncontrolled microorganisms (Lupien, 1997). The Log g (CFU Accepted(CFU g Article -1 ) in12days and the samples were described as not spoiled by 56%of trained judges and 2 has recently been shownto beone of the predominating species in chilled packed food, but - 7%N -1 ). The ). The products inoculated were characterized by buttery andsour odors after 2weeks of L.piscium 2 ) andstored at 6°C. The concentration of both strains reached approximately 8 L. piscium 2 - 50%N hasbeen studied in different food matrixes by inoculating different were inoculated on porkmeat packed under MA conditions (71% O 2 ), theconcentration ofL.piscium L. piscium at and seafood products, the initial number of on CSSor cooked shrimp (Matamoros et al., 2009b; ents (Borch et al., 1996). The microbial selection hasa lightly spoiling effect (Rahkila et al., Photobacterium increased from 3to9Log in seafood (Chaillou Brochothrix 2

100% N studied in bell pepper simulation medium underthree different conditions of gas composition: (i) suggesting that the combination of high O g and 2,3butanedione (diacetyl) after 13 days in the MAP This article isprotected All rights bycopyright. reserved. (70% O diacetyl and acetoin produced by Man-Rogosa-Sharpe (MRS) medium withoutacetate and with 2%glucose, afinal concentration of with thespoilageof food(Vihavainen et al.,2007 storage. Thebuttery off-odor was related to diacetyl/acetoin formation, which is frequently associated 50% CO acetoin/diacetyl and acetate, are produced by these pathways. formate lyase pathways (Andreevskaya etal., 2015). Many significant spoilage substances, such as pyruvate utilization: acetoin/diacetyl, pyruvate dehydrogenase, L-lactate dehydrogenase and pyruvate- results are supported by the presence in the spoilage, such as ethanol after 7 days in 100% N spoiling effect. This effect was correlated to the production of some metabolites that are involved in on L.piscium. Only onestrain, showing the best growth in strain isolated from beef was able to The spoilage effect of three strains of strong rancid odor (personal communication from SouadChristeans, 2014). (lowering 0.45units the pH) anddeteriorated the color, which became gray/green andreleased a to gray (Denis et al., 2014). Inground beef under the same conditions, -1

Accepted) withsome differences in growth sp Article 2 2 2 - 30%CO ; (ii)O air: 21% (Pothakos et al., 2014a). In the first three conditions, all strains reached about 7-9 Log (CFU 2 ) ground veal at 8°C, L.piscium 2 and 79%N L. piscium grow and reached about 8 Log (CFUg 2 L. piscium ; (iii) MAP eed between the strains. For the MAP 2 andCO L. piscium reached 8.5 mM (Andreevskaya etal., 2015).In MAP isolated from beef,pork andsweet peppers was 2 and MAP 1 ; Jääskeläinen et al., 2015). After 48h in modified : 30% CO 2 concentration had asignificant inhibitory effect was shown to modify the color greatly, from red genome of four predicted pathways for for pathways predicted four of genome 2 condition (Pothakos et al., 2014c). These all packing conditions, had asignificant 1 2 conditions, acetic acid after 7 days in air and 70% N L. piscium 2 and (iv)MAP -1 ) atthe endof storage, 2 condition, only the acidifiedmeat the 2 : 50% O 2 and vary according toits interaction with the spoiling microorganisms. batches tested, from different smokehouses, suggesting that the protective effect of this strain may beneficial effect of quality of the same batch of CSS.In VP another set of experiments, Leroi et al. (2015) confirmed the protective effect seems tobe strain-dependent as showed that the strain CNCM I-4031 improved th released very strong off-odors described as “cheese and feet” by sensory panel. These authors also shrimps inoculated with L.piscium demonstrated for the first time by Matamoros et al. (2009a). After 28 days ofstorage at8°C, the This article isprotected All rights bycopyright. reserved. food preservation environment of food have been developed and ha processed and fresh-tasting food.In this context, chilled storage andmodification of the gaseous Protective effect of AcceptedThe positive effect of years, mainly inseafood. Ghanbari et al., 2013). In this context, the role of and Leuconostoc Articlestudies have demonstrated the interest of LABsuch as inhibitundesirable componentsmicrobiota, ofthe an is increasing practice industry. inthefood Many Biopreservation, which consists of inoculating food with selected protective bacterial strains that can development of a wide range ofundesirable microorganisms, like pathogenic and spoilage bacteria. time(Ross etal.,2002).physico-chemical The characteristics products ofthese allowthe safety andquality of the product has to be maintained throughout asignificantly-increased storage During the recent years, consumers have shown a great interest in ready-to-eat, minimally for this purpose (for a review, see Rouse and van Sinderen, 2008; Lacroix, 2010; (Cortesi et al.,2009) L. piscium Lactococcus piscium L. piscium CNCMI-4031 inone batch of naturally contaminated CSSout of two on the sensory quality of cookedVP and peeled tropical shrimp was CNCMI-4031 andEU2229were not spoiled whereas the control . However, the drawback ofthese technologies is that the

L. piscium ve become important and acceptable methods for L. piscium e sensory quality of CSS.However,VP the Carnobacterium EU2229 had no effect onthe sensory has been extensively studied in recent , Lactobacillus , Lactococcus

Lactobacillus B.thermosphacta of inhibition The peeled shrimps (Fall et al., 2010a; 2012) and totally This article isprotected All rights bycopyright. reserved. Antimicrobial activity of Salmonella L. piscium such as B. thermosphacta Pseudomonas piscium Lactobacillus 2009a; Fall et al., 2010a). These strains had a large activity spectrum towards strains of relevant in meat and seafood wastested using adiffusion test on Petri dishes (Matamoros etal., of (Matamoros et al., 2009a; Fall et al., 2010b). The antagonist activity against performed onvarious culture media and food matrix demonstrated that the inhibition of properties has not yet to be elucidated, remaining achallenge for researchers. Different tests 2005). In thecase of L.piscium CNCMI-4031, themechanism involved inits antimicrobial 1988); (iii) hydrogen peroxide (Alomar et al., 2008) and with (iv) nutrient competition (Nilsson et al., reuterin (El-Ziney etal.,1999); (ii) organic acids, such asacetic and lactic acid (Wong andChen, production of (i) antimicrobial peptides, such as bacteriocins (Stiles, 1996; Brillet etal., 2005) or The antimicrobial activities ofLABagainst food spoiling bacteria is generally associated with the in and VP MAP cooked and peeled shrimp by Matamoros et al. (2009a) and Fall et al. (2010b). foodstuffs (GambarinLom etal.,2012; particularly relevant in meat and seafood since this pathogen isfrequently isolated from these

Accepted ArticleL. piscium The antimicrobial activity of CNCMI-4031 inhibited the growth of Carnobacterium is also able to inhibit the growth of pathogens oropportunistic pathogens such as , Staphylococcus aureus, Clostridium sporogenes CNCM I-4031 andEU2229 against Gram-positive and -negative spoilage bacteria , Carnobacterium spp. in meat (Castellano and Vignolo, 2006; Russo et al., 2006) and , which is considered amajor spoilage bacteria in andVP MAP meat andseafood. Serratia spp. in MAP shrimp (Laursen etal., 2006). Lactococcus piscium (Table 3). The inhibitory activity wasconfirmed ona seafood matrix for , Vagococcus, Enterococcus L. piscium had been reported previously withsomesuch as LAB onaco et al., 2015). Its anti-listeria has not been commonly tested. The inhibitory capability B

. thermosphacta stopped its growth in CSS(Leroi et al., 2015). , Psychrobacter andL.monocytogenes by 3-4 (CFU.g Log

activity hasbeen confirmed , Shewanella L. monocytogenes but not with some other (Table 3). -1 ) incooked and Brochothrix , L. E. coli is , , L. sour off-odors. In seafood, some strains can prevent the spoilage and extend the sensory shelf-life. alters the quality ofmeat, with discoloration, acidification and the production of buttery, rancid and most cases, temperatures. The spoiling activity depends on the strain, the food matrix and storage conditions. In Lactococcus various meat, seafood, vegetable and dairy products. It isthe only psychrotrophic species in the genus Conclusion a non-uncharacterized, cell-to-cell contact-dependent that are never been reported in LAB. by L.piscium. diffusion chamber, where bacteria were separated by a filter membrane, nor in medium pre-fermented to nutrient competition (various compounds tested). The inhibition occurred in co-culture but not in a conditions. As an example, H been identified. However, these different gene clusters may notbeactivated in all environmental This article isprotected All rights bycopyright. reserved. putative bacteriocins. In addition, various enzymes putatively involved in H putativeantimicrobial factors. Thisstrain contains th activity but its genome analysis has revealed the presence of some genes that could beinvolved in et al., 2009a; Saraoui et al., 2016). The strain MKFS47 has not been studied for its antimicrobial monocytogenes Accepted Saraoui et al. (2016) demonstrated that the inhibition of inhibition of Articlesubstrates inmilk between For instance, Juillard et al. (1998) showed anutrient competition for non-protein nitrogenous also beenreportedexplain to the competition be glucose under aerobic and anaerobic conditions (Andreevskaya etal., 2015). Nutrient depletion has L. piscium L. piscium and thus can play animportant role in minimally processed products stored at chilled L. monocytogenes These results indicate that the inhibition of was not due to the production of extracellular antimicrobial compounds (Matamoros is gaining the interest of researchers because it is increasingly isolated from does notspoil seafood and even has aprotective effect, whereas it strongly Lactococcus 2 O by Carnobacterium piscicola 2 wasproduced from glycerol under aerobic conditions but not from and Leuconostoc tween microbial populations (Hibbing etal., 2010). ree gene clusters involved in the biosynthesis of of biosynthesis the in involved clusters gene ree L. monocytogenes strains. Nilsson et al. (2005) showed that the L. monocytogenes was due to the competition for glucose. by L.piscium 2 by L.piscium O 2 biosynthesis have was not due is through

Andreevskaya, M., Johansson, P.,Laine, P., Smolander, O.-P., Sonck, M.,Rahkila, R., Jääskeläinen, Alomar, J.,Loubiere, P.,Delbes, C.,Nouaille, S. and Montel, M.C. (2008) Effect of Reference interest. of conflict no is There Conflict ofinterest for her advices. and Research. The authors thank DrDelphine Passerini for assistance with phylogenetic analysis and Taous Saraoui was the recipient of a Ph.D. fellowship from theFrench Ministry ofHigher Education Acknowledgments This article isprotected All rights bycopyright. reserved. Accepted Articleconcerning thespoiling and protective potential of LAB. Comparative genomic and transcriptomic analyses may helptoanswer many questions mechanism still remains unknown. This inhibitory effect of bacteria enumerated by classic and culture-independent methods (Leroi et al., 2015). Ananti-listerial effect observed in one batch ofCSScould not be attr refrigerated packed meat and fish. However, other mechanisms are probably involved, asaprotective This has been attributed to the inhibition of

E., Paulin, L.,Auvinen, P. and Björkroth, J. (2015) Genome sequence and transcriptome aureus garvieae, Lactococcus lactis L. piscium inmicrofiltered milk. has also beenobserved andattributed to cell-to-cell contact, although the and Enterococcus faecalis Food Microbiol B. thermosphacta mechanism hasnever been described in other L. piscium ibuted to the reduction of this species, nor other 25 , 502–508. . , amajor spoilage bacterium in onthe behaviour of Staphylococcus Lactococcus Cavanagh, D.,Fitzgerald, G.F. andMcAuliffe, O.(2 Castellano, P. and Vignolo, G.(2006) Inhibition of Listeria innocua Carraro, L., Maifreni, M., Bartolomeoli, I., Martino, M.E., Novelli, E.,Frigo, F., Marino, M. and Brillet, A.,Pilet, M.-F., Prevost, H.,Cardinal, M. and Leroi, F.(2005) Effect of inoculation of Boucher, I., Vadeboncoeur, C.andMoineau, S. (2003) Characterization of genes involved inthe Borch, E., Kant-Muermans, M.L.and Blixt, Y. ( Bolotin, A., Wincker, P.,Mauger, S., Jaillon, O.,Malarme, Weissenbach, K., Ehrlich, J., S.D. and Björkroth, Ristiniemi, J., M., Vandamme, P.and Korkeala, H. (2005) Cai, Y.,Yang, Pang, J., H.and Kitahara, M. (2011) This article isprotected All rights bycopyright. reserved. Accepted Article Lactococcus lactis and its bacteriocins. Lett App Microbiol in vacuum-packaged meat by addition of bacteriocinogenic communitymonitoring during Montasio cheese manufacturing. Cardazzo, B.(2011) Comparison of culture-depe 267-276. Carnobacterium divergens V41, a biopreservative strain against 4056. metabolism ofα products. lactis Sorokin, A.(2001) The complete genome sequence of the lactic acid bacterium Carnobacterium dominating in fresh modified-atmosphere-packaged, marinated broiler legs are overgrown by Microbiol analysis ofmeat spoilage lactic acid bacterium Microbiol on themicrobiological, chemical andsensory quality of cold-smoked salmon. bacterium isolated from vegetable matter. ssp. Int JFood Microbiol 33 lactis 81, 3800–3811. 104, 309-324. IL1403. GenomeRes -Galactosides by and Lactobacillus and its domestication to the dairy environment. , 103-120. Lactococcus raffinolactis Lactococcus species during storage at6°C. 11, 731-753. 1996) Bacterial spoilage of meat and cured meat 43, 194-199. Int JSyst Evol Microbiol Lactococcus fujiensis 015)From field tofermentation: origins The of Lactococcus piscium Lactococcus ndent and-independent methods for bacterial . AppEnviron Microbiol 69,4049- Lactobacillus curvatus and Enterococcu Res Microbiol 162, 231-239. Listeria monocytogenes Brochothrix thermosphacta sp. nov., alactic acid Int JFood Microbiol Food Microbiol 61 MKFS47.App Environ , 1590-1594. s species Int J Food Int J Lactococcus CRL705 47,45-61. 97, risk,

This article isprotected All rights bycopyright. reserved. Chen, Y.-s., Chang,-h., Pan,S.-f., C. Wang, L.-t., Chang,-c., Y. Wu,H.-c. andYanagida, F. Chen, Y.,Otoguro, M., Lin, Y.,Pan,S., Ji, S.,Yu, Chaillou, S., Chaulot-Talmon, A.,Caekebeke, H.,Card Chaillou, S., Chaulot-Talmon, A.,Caekebeke, H.,Cardinal, M., Christieans, S., Denis, C.,Hélène Cho, S.-L., Nam, S.-W., Yoon, J.-H., Lee, J.-S., Sukhoom, A.andKim, W.(2008) Cortesi, M.L., Panebianco, A., Giuffrida, A., AcceptedDaniel, C.,Sebbane, F., Poiret, S., Goudercourt, D., Dalgaard, P. (1995) Qualitative and quantitative characterization of spoilage bacteria from packed Article cummingcordia. (2013) (fermented broccoli stems). Int J Syst Evol Microbiol (2014) /v9/n5/full/ismej2014202a.html http://www.nature.com/ismej/journal/v9/n5/suppinfo/ismej2014202s1.html?url=/ismej/journal and Champomier-Vergès, M.-C. (2015b)supporting-files-isme2015-202-v5.0. 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(1997)TheandHebraud,badie, M. coldshock J. ick, E.,Low,D.A.,Hayes, C.S.,Goulding, C.W. , Y., Ritzenthaler, P.,Daveran-Mingot, M.-L. and is, A.,DeMeulenaer, B.and Devlieghere, F. Lactococcus garvieae Lactococcus , 172-183. involvesfour low-molecular- by anonbacteriocinogenic . PLoSONE Leuconostoc 6(8).

Sarika, A.R.,Lipton, A.P., Aishwarya, M.S. and Saraoui, Fall,T., P.A.,Leroi, F.,Antignac, J.-P Samarzija, D.,Antunac, N. andHavranek, J.-L. Taxonomy, (2001) physiologygrowth of and Sakala, R.M.,Hayashidani, H.,Kato, Y., Kaneuchi, C. and Ogawa, M.(2002b) Isolation and Sakala, R.M.,Hayashidani, H.,Kato,Y.,Hira Russo, F.,Ercolini, D., Mauriello, G.and Villani, F.(2006) Behaviour of Brochothrix thermosphacta Rouse, S.and van Sinderen, D.(2008) Bioprotective Potential of Lactic Acid Bacteria in Malting and Ross, R.P., Morgan, S. and Hill, C. (2002) Preservation andfermentation: past, present and future. Rahkila, R.,Nieminen, T., Johansson, P., Säde,E. This article isprotected All rights bycopyright. reserved. Accepted Article 1280-1289. producing Lactococcus lactis high-value marine fish under different storage temperatures. CNCM I-4031. mechanism ofListeria monocytogenes by abioprotective bacteria Lactococcus lactis App Microbiol characterization of Lactococcus piscium vacuum-packaged beefduring chiller storage. Kaneuchi, C.andOgawa, M.(2002a) Change inthe composition ofthe microflora on in presence of other meat spoilage microbial groups. Brewing. Brewing. atmosphere packaged meat. of potential spoilage the of evaluation simulation medium underdifferent gas compositions. gelidum J Food Microbiol subsp. J FoodProt 92, 173-179. gasicomitatum gasicomitatum Food Microbiol 53,70-78. 79, 3-16. : a review. Mljekarstvo51 71, 1724-1733. Int J Food MicrobiolInt J and application of Its bacteriocin to manage spoilage bacteria in and Lactococcus piscium Lactococcus piscium ., Chéreau,S.and Pilet, M. F.(2016) Inhibition strains from vacuum-packaged refrigerated beef. Dhivya, R.S.(2012)Isolation of aBacteriocin- ta, T., Makino,ta, T., Y.,Fukushima,A., Yamada, T., and Björkroth, J. (2012) Characterization and , 35-48. Int JFood Microbiol 74,87-99. 156, 50–59. Food Microbiol 23,797-802. Int JFood Microbiol , onsweet bell pepper (SBP) isolates from modified AppBiochem Biotechnol 167 Lactococcus piscium 178, 120-129.

, J Int Wong, H.-C. andChen,Y.-L. (1988) Effects of Lact Williams, A.M.,Fryer, J.L., Collins and M.D. (1990) Von Wright, A.(2012) Genus Vihavainen, E.,Lundström, H.-S., Susiluoto, T., Koort, Paulin, J., L.,Auvinen, P. and Björkroth, K.J. andPapadimitriou, K. (2011) Stre Tsakalidou, E. Stiles, M.E.and Holzapfel, W.H. (1997) Lactic acid bacteria of foods and their current taxonomy. Stiles, M.E.(1996) Biopreservation by lactic acid bacteria. Vendrell, D.,Balcázar, J.L., Ruiz-Zarzuela, I., de This article isprotected All rights bycopyright. reserved. Accepted Article Schleifer, Kraus, J.,Dvorak, K.H., Kilpper-BälzC., and Germination of Bacillus cereus. Lactococcus Raton: CRC Press. CRCPress. Raton: Aspects ed.Lahtinen, S.,Ouwehand, A.-C., Salminen,S. andvon Wrigh, A.,pp.63-76. Boca Microbiol atmosphere-packaged broiler products with psychrotrophic lactic acid bacteria. (2007) Role ofbroiler carcasses and processing plant air in contamination of modified- Springer Science & Business Food Microbiol 36,1-29. AppMicrobiol Lactococcus garvieae of Streptococcus lactis and related 73, 1136-1145. species from salmonid fish. 6, 183-195.- Lactococcus in fish: A review. Media. , InLactic Acid Bacteria: Microbiological and Functional Streptococci App Environ Microbiol Comp ImmunolMicrobiol Dis Infect Blas, I., Gironés,O.and Múzquiz, (2006) J.L. FEMS MicrobiolLett ss Responses of Lactic Acid Bacteria. London: , R.,Collins, M.D. and Fischer, W.(1985)Transfer ic Acid Bacteria andOrganic Acids on Growth Lactococcuspiscium to thegenus Antonie vanLeeuwenhoek 70,331-345. 54, 2179-2184. Lactococcus 68, 109-113. sp. nov. anew gen. nov. Syst 29 App Environ , 177-198.

Int J in Elliker medium (x30000). Figure 2: Scanning Electron Microscopy of outgroup species. This article isprotected All rights bycopyright. reserved. Accepted Articleneighbor-joining method withbootstrap of 1000 replicates. W software. The construction of phylogenetic trees was performed with MEGA 6 Toolbar using the After thetrimming gapsof the andmissing data,atotal of1330positions werealigned using clustal two genome sequenced L. ( piscium sequences of the eleven different Phylogenetic relationship of species andsubspecies of thegenus Figure 1 Lactococcus ♦ ), were retrieved from GenBank (http://www.ncbi.nlm.nih.gov). Lactococcus piscium species, including the four subspecies of L. lactisof subspecies four the including species, Lactobacillus curvatus CNCMI-4031after 24 hof culture Lactococcus . The 16SrRNAgene was used as an and Meat

EU2229

Fish and sellfish condition and storage Food This article is protected All rights bycopyright. reserved. Table 1: Food sources of - - - Accepted- - Article- Raw salmon-MAP 4-8°C MAP meet product MAP fresh salmon-8°C VP beef VP beef - 2°C Fresh VP Rainbow trout fish

Lactococcus piscium

MIP 2434, MIP 2450, MIP 2482, MIP 2484 Not spoiled/Lightly spoiled (raw salmon) salmon) (raw spoiled spoiled/Lightly Not 2484 MIP 2482, MIP 2450, MIP 2434, MIP

LMT33-6 4031 I CNCM C2T15 C2T11, A2T2, E2B2, R-46592 GTC 552 Strain Strain and its sensory effect, when inoculated into fish and meat product. Butter and fatty fish - 12 days ofstorage week feet of storage - 4 and Cheese Not spoiled (shrimp) 4 week of storage sour odors- and acid, amine, Cheese/feet, Butter and smoke odors - 2 week of storage Not spoiled/spoiled (cold smoked salmon) Cheese and feet/no odors - 4 week of storage Not spoiled (shrimp) Buttery- 14 days Lightly spoiled (pork) Butter and smoke odors - 4 week of storage Not spoiled (cold smoked salmon) Not examined examined Not Spoiler (sweet ball pepper simulation medium) Not examined Sensory effect effect Sensory

Macé et al. (2013) (2013) al. et Macé Matamoros et al., (2009a), (2009b) (2009b) (2009a), al. et (Matamoros Matamoros et al., (2009a), (2009b) Fall et al. (2010a), (2012); Rahkila et al.(2012) Matamoros et al., (2009a), (2009b) Pothakos et al.(2014a), (2014c) Williams et al., (1990)

Sakala et al. (2002b) (2002b) al. et Sakala Reference Reference Vegetable Vegetable JL3-4 This article is protected All rights bycopyright. reserved. - - - Accepted - Article A rie ie tis MKFS47 MAP broiler filet strips porkVP VP porkVP - 4°C Sweet bell pepper salad (air)

EU621998 R-46738

R-46976 Lightly spoiler (sweet ball pepper simulation medium) Buttery Lightly spoiler (sweet ball pepper simulation medium) Not examined Sour – 16 days Lightly spoiled (pork) Spoiled (pork) (2015) al. et Andreevskaya (2010) al., et Jiang Rahkila et al. (2012) Pothakos et al.(2014b) Pothakos et al.(2014a), (2014c) CAU 28 Lactococcus chungangensis 16395 Lactococcus fujiensis JCM 4877 Lactococcus raffinolactis ATCC 49156 garvieae Lactococcus cremoris lactis Lactococcus lactis lactis Lactococcus I-4031 MKFS47 piscium Lactococcus piscium Lactococcus Strain Genome

This article is protected All rights bycopyright. reserved. Table 2: Genome overview of some Accepted IL1403 Article

SK11 subsp. subsp. CNCM number (GenBank) number (GenBank) BL0000128 3. n 25 4 47 http://www.ncbi.nlm.nih.gov 4 2252 nd 36.9 2088 BBAL00000000.1 CALL00000000 2280 38.7 0 2418 - 48 Meslier et al. (2012) Meslier et al. (2012) 48 - 2418 0 38.7 2280 CALL00000000 LN774769- In progress 2257 39 1 2239 4 55 Marché et al. (2014) et al. (2014) Marché 55 4 2239 1 39 2257 In progress AE005176 AP009332 1950 38.8 0 1947 5 62 Morita et al., (2011) Morita et al., (2011) 62 5 1947 0 38.8 1950 AP009332 CP000430 LN77477 Lactococcus -

deposit strains genome sequenced including the two size (kbp) size Genome 2641 35.8 5 2509 6 62 Makarova et al., (2006) et al., (2006) Makarova 62 Bolotin et al. (2001) 62 6 2509 6 5 2310 35.8 0 2641 35.4 2365 2394 38.79 2 2476 4 56 Andreevskaya et al., (2015) al., (2015) et Andreevskaya 56 4 2476 2 38.79 2394 23 0 d 14 4 Cho et al. (2008); 47 3 2194 nd 40 2243 GC% Plasmid(s) CDS Ribosomal Ribosomal CDS Plasmid(s) GC% L. piscium operons operons strains genome sequenced. RNA tRNA Reference Reference tRNA

https://www.patricbrc.org https://www.patricbrc.org

supernatant (MSMA medium) (MSMA supernatant with assay spot Agar

Cold smoked salmon AcceptedThis article is protected All rights bycopyright. reserved. shrimp cooked and Peeled Co-culture medium on MSMA Article L. piscium with assay spot Agar Methods Table 3: Antimicrobial activity of

L. piscium

colony colony Lactococcus piscium Lactococcus CNCM I-4031 CNCM I-4031 CNCM I-4031 CNCM I-4031 CNCM I-4031 strains piscium L. EU2229 Target Target strain strains against spoilage and pathogenic bacteria relevant in food Listeria monocytogenes monocytogenes Listeria maltaromaticum Bacillus subtilis Typhimirium, Serratia liquefaciens Brochothrix thermosphacta Staphylococcus aureus enterica Shewanella putrefaciens Pseudomonas Listeria monocytogenes monocytogenes Listeria Psychrobacter farciminis , Photobacterium phosphoreum Brochotrix. thermosphacta monocytogenes Listeria Brochothrix thermosphacta monocytogenes Listeria Bacillus subtilis Staphylococcus aureus

Listeria monocytogenes Listeria sp., sp., sp., Pseudomonas sp., , , Staphylococcus xylosus , Clostridium sporogenes Staphylococcusxylosus Serratia liquefaciens Vagococcus fluvialis , Vagococcus , Brochothrix thermosphacta

, Clostridium sporogenes ,

, Carnobacterium alterfunditum Serratia proteamaculans sp., Salmonella enterica , , Serratia Photobacterium phosphoreum, , Photobacterium phosphoreum , Vibriosp Escherichia coli , Escherichia Vagococcus carniphilus carniphilus Vagococcus and , sp., Escherichia coli Escherichia Shewanella putrefaciens , Lactobacillus farciminis .

, Psychrobacter Psychrobacter serovar , Lactobacillus , C. divergens , sp., Salmonella ,

, C. , and o niie Fall et al. (2010a); Saraoui Not Inhibited Fall et al. (2010a); Not Inhibited Growth Growth niie Fall et al. (2010a); Inhibited o niie Matamoros et al. (2009a) Not inhibited niie Matamoros et al. (2009a) Inhibited o niie Leroi et al. (2015) Fall et al. (2010b) Not inhibited Saraoui et al. (2016) Inhibited Inhibited niie Fall et al. (2010a) Inhibited niie Leroi et al. (2015) Inhibited inhibition et al. (2016) (2016) al. et (2009a) al. et Matamoros Matamoros et al. (2009a) (2009a) al. et Matamoros Reference Reference This article isprotected All rights bycopyright. reserved. Accepted Article

This article isprotected bycopyri Accepted Article g g ht. Allrights reserved.