CHARACTERISATION, IDENTIFICATION AND TECHNOLOGICAL PROPERTIES OF PSYCHOTROPHIC LACTIC ACID BACTERIA ORIGINATING FROM TUNISIAN FRESH FISH

CHARACTERISATION, IDENTIFICATION AND TECHNOLOGICALPROPERTIES OF PSYCHOTROPHIC LACTIC ACID BACTERIAORIGINATING FROM TUNISIAN FRESH FISHjfs_385 1..12

MOUNA BOULARES1,3, CHEDIA AOUADHI2, MELIKA MANKAI1, OLFA BEN MOUSSA1, INES ESSID1 andMNASSER HASSOUNA1

1Unité de Recherche “Sciences et Technologies des Aliments”, École Supérieure des Industries Alimentaires de Tunis (ESIAT), 58 Avenue Alain Savary,Cité El Khadhra, Tunis 1003, Tunisie2Laboratoire de Microbiologie, “Groupe bioprocédés”, Institut Pasteur de Tunis (IPT), Belvédère, Tunis, Tunisie

3Corresponding author. TEL: +21620323405;FAX: +21671771192; EMAIL:[email protected]

Received for Publication January 9, 2012Accepted for Publication May 22, 2012

doi:10.1111/j.1745-4565.2012.00385.x

ABSTRACT

One hundred sixty psychrotrophic lactic acid bacteria (LAB) isolated from wild andaquacultured fresh fish were identified by biochemical and molecular methods using16S-23S rRNA spacer analysis and 16S rDNA sequencing. LAB strains were charac-terised according to their technological properties including acidifying capacity,antibiotic resistance and proteolytic, lipolytic and enzymatic activities, as well asantimicrobial activities, against pathogenic and spoilage bacteria in order to selectthe most suitable strains for use as starter cultures in the biopreservation of freshfish and seafood products. The majority of strains displayed antimicrobial activitiesagainst pathogenic bacteria (Salmonella arizonae, Staphylococcus aureus, Listeriamonocytogenes, Pseudomonas aeruginosa, Escherichia coli and Aspergillus flavus) andthe dominant psychrotrophic Gram-negative bacteria in fresh fish (Pseudomonasfluorescens, Aeromonas hydrophila, Pseudomonas putida and Photobacterium damse-lae). A total of 39 isolates of LAB isolates having the most important antimicrobialactivities were selected for further properties. Low proteolytic and lipolytic activitieswere detected for all strains using azocasein methods and fish fat, respectively.Moreover, all strains showed acidifying activity by reducing pH to less than 4.38 at37C after 72 h. Finally, all studied LAB were resistant to penicillin, gentamicin,colistin, ciprofloxacin, norfloxacin, nalidixic acid, erythromycin, tetracycline andstreptomycin.

PRACTICAL APPLICATIONS

The paper presents the identification and the technological properties of lactic acidbacteria (LAB) including antimicrobial activity against pathogenic and spoilage bac-teria in order to select the most suitable strains for use as starter cultures in the bio-preservation of fresh fish and seafood products. Considering this, assessment of theeffect of inoculation of selected LAB on microbiological and physicochemical prop-erties of fresh fish stored at chilled temperature is necessary to guarantee adequatefresh seafood products safety. Therefore, these analyses can lead to develop betterbiopreservation strategy for fresh fish by the use of selected LAB strains.

INTRODUCTION

Fresh fish is an extremely perishable proteinaceous food thatspoils due to the metabolism of spoilage microbiota whichends up in foods because of cross contamination (Gram and

Huss 1996; Boularès et al. 2011a). Various food preservationtechniques have been utilized to improve the microbial safetyand extend the shelf life of fish in general, including freezing,dehydration, fermentation (Papamaloni et al. 2003; Adolpheet al. 2006; Roberts et al. 2008), chemical preservation, salting

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Journal of Food Safety ISSN 1745-4565

1Journal of Food Safety •• (2012) ••–•• © 2012 Wiley Periodicals, Inc.

and smoking (Glatman et al.2000; Gelman et al.2001; Nickel-son et al. 2001; O’Sullivan et al. 2002; Ligia et al. 2008; Dortuand Thonart 2009). Among the wide array of strategies beingcurrently used or proposed for food preservation, controlstrategies based on living organisms and/or their antimicro-bial products have been used for centuries and are becomingincreasingly popular for several reasons (Ashmaig et al. 2009;Galvez et al.2010): natural preservation methods are regardedashealth friendlybyconsumersandtheymaydecrease thepro-cessing costs while at the same time extending the productshelf life period. This circumstance has increased interest inresearch on antibacterial and antifungal agents such lactic acidbacteria (LAB) and LAB bacteriocins because they can be rec-ognized as safe compounds (Kabadjova et al. 2002; Alves et al.2006; Abdelbasset and Djamila 2008; Ananou et al. 2010) indairy, vegetable, fish, seafood and meat products (Matamoroset al. 2006; Aymerich et al. 2008; Najjari et al. 2008). Prolifera-tion of LAB often found on skin, gills and in the gastrointesti-nal tract of fish products (Ringo and Gatesoupe 1998) canprevent the development of undesired bacteria and inactivatepathogens (Diop et al.2010; Boularès et al.2011a),and thanksto their ability to produce antimicrobial compounds such aslactic acid, acetic acid, diacetyl, acetoin, hydrogen peroxide,reuterin,antifungal peptides (Aymerich et al.2000) and bacte-riocins (Ligia et al. 2008; Leroi 2009; Diop et al. 2010). More-over, in food products, LAB cause a decrease in pH (generallybelow 5–4.5) which (1) improves the product safety, stabilityand shelf life by inhibiting undesirable changes brought aboutby spoilage and pathogen microorganisms or abiotic reactionsthat favour product safety by inactivating (Essid et al. 2009)and (2) creates the biochemical conditions to attain the finalsensory properties, aroma, texture developments and appear-ance (Kleerebezemab et al. 2000; Galvez et al. 2007; Ashmaiget al. 2009) through modification of the raw materials. InTunisia, several food products derived from meat or seafoodare naturally processed, without any addition of bacterialstarters,such as fermented and salted,as well as the raw meat orfish (Najjari et al. 2008). For this reason, the aim of thisresearch was to identify and characterise the predominant psy-chrotrophic LAB isolated from fresh fish. The second purposewas the selection of the most suitable strains according to theirtechnological characteristics including acidification, enzy-matic activities and antibiotic resistance and to their safetycharacteristics including antagonistic activity against spoilagestains and foodborne pathogens to be used as starter culturesfor preservation of fish.

MATERIALS AND METHODS

Isolation of LAB Strains

Psychrotrophic LAB strains, used in this work, were isolatedfrom 80 samples of 12 species of wild and aquacultured fresh

fish (sea bass, sea bream, mullet, whiting, sole, sardine, mack-erel, red mullet, octopus, shrimp, cuttlefish and pageot).These fish samples have been provided from two markets ofTunisia, packed in cold boxes and transferred to the labora-tory within 1 h for analyses (Boularès et al. 2011a). Uponarrival, fish were immediately gutted, headed, washed and fil-leted. Ten grams of muscle obtained from each fish specieswas transferred aseptically to a Stomacher bag containing90 mL of peptone water (Biokar Diagnostics, Bauvais,France) and homogenized for 3 min using a laboratoryblender Stomacher (BagMixer, Saint Nom La Bretèche,France).Then,decimal progressive dilutions were carried out.

Strains of psychotropic LAB were isolated and purified onMan Rogosa and Sharpe (MRS) agar (Pronadisa, Madrid,Spain) and M17 agar (Pronadisa) after incubation at 7Cduring 10 days (Guiraud 1998; Boularès et al. 2011a,b).

Phenotypic Characterisation

Morphology, Gram staining, catalase and oxidase tests,mobility, oxide fermentation and oxygen tolerance werecarried out. All isolates were checked for gas production fromglucose in MRS broth (Pronadisa) containing Durham tubes(Greco et al. 2005). Tryptophanase activity was detected withKovac’s reagent as described by Guiraud (1998). Only Gram-positive, catalase negative strains were considered duringfurther analyses. Then, carbohydrate fermentation patternsof LAB isolates were determined using the API 50 CHL system(BioMérieux, Craponne, France). The computer programAPIWEB database identification software (BioMérieux) wasused to interpret the results. Strains were stored at -20C inMRS broth containing 20% glycerol.

Technological Properties of LAB StrainsIsolated from Fish

Antimicrobial Activity. Agar well diffusion assay was usedfor detection of antagonistic activity as described by Papama-loni et al. (2003), Ben Moussa et al. (2008) and Essid et al.(2009). Twenty millilitres of Nutrient agar (Biokar Diagnos-tics) was inoculated in a Petri dish with 100 mL of an overnightculture of the indicator microorganisms (Pseudomonas fluore-scens, Pseudomonas putida, Aeromonas hydrophila and Photo-bacterium damselae). Then, wells of 5 mm in diameter wereperforated and 15 mL of an overnight culture of the LAB strainwas loaded in wells. Subsequently, each antagonistic activitywas related to the area (square millimetre) of the clear zoneobserved surrounding wells after incubation. Strains havingthe most antibacterial activities were then tested for their anti-bacterial and antifungal activities against six undesirablestrains: Listeria monocytogenes,Salmonella arizonae,Staphylo-coccus aureus, Pseudomonas aeruginosa, Escherichia coli (DH5alpha, Institute Pasteur of Tunisia) and Aspergillus flavus.

LACTIC BACTERIA M. BOULARES ET AL.

2 Journal of Food Safety •• (2012) ••–•• © 2012 Wiley Periodicals, Inc.

Proteolytic Activities. Initially, surface-dried plates ofskimmed milk agar (Thapa et al. 2006; Ben Moussa et al.2008) and gelatine agar were streaked with an overnightculture of each strain (MRS broth, 24 h, 37C). After incuba-tion, the proteolysis was determined by measurement of thearea (square millimetre) of the clear zone surrounding theinoculated spots. Also, the proteolytic activity assay was per-formed with selected strains by the absorbance measured at440 nm in UV/visible spectrophotometer (Jenway 6305 spec-trophotometer), using azocasein as substrate, as described byThapa et al. (2006) and Essid et al. (2007).

Lipolytic Activities. First, the production of extracellularlipases and extracellular phospholipids (lecithinase) wasdetermined, respectively, on nutritive agar plates that con-tained 1% Tween 20,1% Tween 80 and Tributyrin as describedabove (Guiraud 1998) and on egg yolk agar as described byDogan and Boor (2003). Lipolytic activity was shown by theappearance of precipitated zone around and under eachcolony in plates containing Tween (Munsch-Alatassova andAlatassova 2005) or opaque ring surrounding lecithinase-positivecoloniesafter2daysof incubation.Moreover,lipolyticactivity was measured by a modified titration method,accord-ing to Mauriello et al. (2004) and Essid et al. (2007) usingcod-liver oil as fish fat (Santiveri, Palma de Mallorca, Spain).

Each lipolytic and proteolytic activity test was performedin duplicate.

Enzymatic Activity. Enzymatic activities were assayedusing the API-ZYM galleries (BioMérieux) as described bythe manufacturer. Each strain was grown on MRS agar platesat 37C for 24 h. Colonies were then removed from the mediaand resuspended in 2 mL of sterile distilled water. The cellsuspension from each strain was used to inoculate the cupulesof the API-ZYM strips. The latter were then incubated 37C for4 h. The reaction was terminated by the addition of the API-ZYM reagents (ZYM A and ZYM B). Enzymatic activity wasgraded from 1 to 5 according to the colour reaction chart. Theapproximate number of free nanomole hydrolyzed substratewas obtained from the colour intensity: 0: no activity; 1:liberation of 5 nmol; 2: 10 nmol; 3: 20 nmol; 4: 30 nmol; and5: �40 nmol (Papamaloni et al. 2002; Essid et al. 2009).

Antibiotic Susceptibility. Susceptibility to antibiotics wastested using a disc diffusion test as described by Ben Moussaet al. (2008). According to the breakpoints recommended bythe National Committee for Clinical Laboratory Standards(CA-SFM 2005), results were expressed as sensitive (S), mod-erate (M) or resistant (R) upon the diameters of inhibitionzone obtained. The isolates were screened for their suscepti-bility to penicillin G (10 UI), ampicillin (10 mg), cefuroxim(30 mg), ceftriaxon (30 mg), gentamycin (500 mg), amikacin(30 mg), chloramphenicol (30 mg), colistin (50 mg), nitro-

furantoin (300 mg), nalidixic acid (30 mg), ciprofloxacin(5 mg), norfloxacin (5 mg), rifampicin (30 mg), erythromycin(15 UI), tetracycline (30 UI) and streptomycin (10 UI). Themultiple antibiotic resistance (MAR) index of the isolates wasdone as referred to Snoussi et al. (2010). The MAR index wasdefined as a/b where a represents the number of multipleantibiotics to which the particular isolate is resistant and b asthe number of multiple antibiotics to which the particularisolates were exposed.

Acidification Activity. pH values of the culture in MRSmediumwererecordedafterusingapHmeter(WTWportablepH-meter pH 315i/SET, Wissenschaftlich). A defined volumeof an overnight culture of each strain (MRS broth at 37C for24 h) was used to inoculate 100 mL of MRS broth to obtain anoptic density at 600 nm (OD600 nm) of 0.8.pH,which was deter-mined after 4, 8, 24, 48 and 72 h of incubation at 4 and 37C.

Growth Rate at Different Temperatures, SaltConcentrations and pH. Growth at different tempera-tures (4, 15, 37 and 45C) and at different pH (3.9, 4.6, 6.5 and9.6), as well as the ability to grow in different concentrationsof NaCl (0.5, 6.5, 10 and 18% [w/v]) in MRS broth, was deter-mined, as described by Schillinger and Lucke (1987), Dykeset al. (1994) and Thapa et al. (2006). One millilitre of an over-night culture of each strain (OD600 nm = 0.8) was inoculated in10 mL of the different media described above and growth wasevaluated by the difference between the OD at time 0 and after24-h incubation at 37C.

Genotypic Characterisation of LAB Strains

Genomic DNA Extraction and Polymerase ChainReaction Amplification. DNA extraction was carried outusing a modified genomic DNA isolation protocol asdescribed by Vaquero et al. (2004) and Ashmaig et al. (2009)using lysosyme and proteinase K (Invitrogen, Carlsbad, CA).Therefore, genotypic identification of selected isolates wasperformed using 16S/23S ribosomal RNA (rRNA) primers(Invitrogen) 16S 5′→3′ GCTGGATCACCTCCTTTC, 23S5′→3′ AGTGCCAAGGCATCCACC (Ben Moussa et al.2008). Chromosomal DNA used for polymerase chain reac-tion (PCR) amplification was prepared according to Ander-son and Mckay (1983) and Ben Moussa et al. (2008) method.

Sequencing. The identification of selected isolates wasconfirmed by sequencing 16S rRNA genes after PCR amplifi-cation, using the universal primers Fd1 and Rd1 (Fd1,5′-AGAGTTTGATCCTGGCTCAG-3′ and Rd1, 5′-AAGGAGGTGATCCAGCC-3′) (Invitrogen), as described by Winkerand Woese (1991). Then, the PCR mixture was subjected to30 cycles as described by Hedi et al. (2009). All sequencingreactions were performed in “Genomic and Biomedical

M. BOULARES ET AL. LACTIC BACTERIA


Ontogenetic Laboratory” in “Pasteur Institute of Tunisia.”Therefore, obtained sequences were compared with availabledatabases using the GenBank BLASTN (Ewing and Green1998; Gordon et al. 1998).

Statistical Analysis. Statistical analyses were performedusing SPSS version 17 software (SPSS, Chicago, IL) to deter-mine differences between means. Statistical significance fordifferences was determined at 5% probability level.

RESULTS

Identification of Isolates

A total of 360 strains were isolated from the samples of wildand aquacultured fresh fish. According to their positive Gramreaction and absence of catalase and oxidase activities, allstrains were considered to be LAB, then, 160 strains werespecified by the API 50 CHL system (BioMérieux). Based onphenotypic characterisation and interpretation of APIWEBdatabase, 22.7% of these strains were identified as Lactococcuslactis (36 strains), 20.57% belonged to the species Lactobacil-lus plantarum (32 strains), 17% as Lactobacillus brevis (28strains), 16.31% as Leuconostoc mesenteroides (27 strains),12.06% as Lactobacillus paracasei (21 strains), also 3.55% asCarnobacterium piscicola (5 strains), 2.84% as Leuconostoccitreum (4 strains), 2.13% as Lactobacillus acidophilus (3strains) and finally 2.84% were identified Carnobacteriumdivergens, Lactobacillus curvatus, Pediococcus pentosaceusand Lactobacillus delbrueckii (1 strain for each species). Themost frequent genera of psychrotrophic LAB were identifiedas belonging to the Lactobacillus, Lactococcus, Leuconostoc,Carnobacterium and Pediococcus. These strains showeddifferences in fermentation abilities of some sugars.

Technological Properties

Antibacterial Activity. One hundred sixty psychrotrophicLAB strains were screened for exhibition of antagonisticactivities against the dominant psychrotrophic Gram-negative microorganisms, isolated from the same fresh fishsamples and possessed the highest proteolytic and lipolyticactivities. All LAB strains were shown to produce inhibitionzones against these dominant spoilage bacteria in freshfish (P. fluorescens, A. hydrophila, P. putida and Ph. damselae).Besides, LAB strains had inhibition capacity against patho-genic bacteria (S. arizonae, St. aureus, P. aeruginosa, E. coliand A. flavus). Based on the obtained results, only 39 strainshaving the most important antimicrobial activity and belong-ing to the most dominant species in fresh fish were selectedfor further technological properties. Furthermore, the 39selected strains belonged to Lc. lactis (strains Lc1 to Lc8),Lb. plantarum (strains Lp1 to Lp4), Lb. paracasei (strains Lpc1

to Lpc4), Lb. brevis (strains Lb1 to Lb6), L. mesenteroides(strains Lm1 to Lm13) and C. piscicola (C1 to C4). Inhibitoryspectra of these strains against psychrotrophic Gram-negative bacteria and pathogenic bacteria are representedin Table 1.

Proteolytic Activity. Proteolytic activity of the 39 selectedLAB strains from the 160 strains isolated from fresh fish,as assessed by the agar well-diffusing method, showed thatthe majority of strains could hydrolyze caseins (Table 2),whereas no result was given on gelatine agar. Strains Lb. plan-tarum (Lp4), Lc. lactis (Lc6), L. mesenteroides (Lm3) andLb. paracasei (Lpc2) showed the highest diameter haloof degradation with the agar plate method (377.8, 319.7,291.4 and 247.4 mm2, respectively). Strains Lc. lactis (Lc1),L. mesenteroides (Lm12) and Lb. brevis (Lb4) had no halossurrounding wells. However, the extracellular proteolyticactivity on azocasein substrate was very poor for all LABstrains (Table 3). The highest ODs at 440 nm (0.855, 0.493and 0.486) were detected for Lp1, Lpc2 and Lp2, respectively.

Lipolytic Activity. Results concerning lipolytic activity onagar mediums supplemented with different fat sourcesshowed that none of the strains was able to hydrolyze Tween20, Tween 80 or tributyrin. All strains do not have lecithinaseactivity neither lipolytic activity. Otherwise, lipolytic activitymeasured using fish fat detected very weak lipolytic capacitywith very close values for all LAB strains (Table 2). Thehighest values (9.52, 8.46 and 7.05) were obtained for thestrains Lp2, Lb1 and Lb4, respectively.

Enzymatic Activities. The enzymatic activities of theselected LAB strains, as evaluated by the semiquantitativeAPI-ZYM system, are shown in Table 3. High leucine aryla-midase and valine arylamidase activities were observed for allstrains, but only two strains of L. mesenteroides (Lm1 andLm8) showed a weak or absent activity. However, cystine-arylamidase activity was low (around 10 and 20 nmol hydro-lyzed substrate). The lipolytic enzyme, phosphatase alkaline,was not detected for the majority of tested strains; in contrast,acid phosphatase activity was present for all strains. Definiteesterase (C4), esterase-lipase (C8) and lipase (C14) activitieswere very weak (around 5 nmol of hydrolyzed substrate)or absent for most strains. On the other hand, most strainshave naphthol-As-Bi-phosphohydrolase and N-acetyl-b-glucosaminidase activities between 10 and 20 nmol,while a-glucosidase and b-glucosidase were approximatelyhigh activities (between 20 and 40 nmol of hydrolyzedsubstrate).

Antibiotic Resistance. Antibiotic resistance results of the39 LAB tested against 16 different types of antimicrobialagents, using the disk diffusion method, were shown in



TAB

LE1.

AN

TIM

ICRO

BIA

LA

CTI

VIT

YO

F39

SELE

CTE

DST

RAIN

SO

FPS

YC

HRO

TRO

PHIC

LAC

TIC

AC

IDBA

CTE

RIA

AG

AIN

STPA

THO

GEN

ICA

ND

UN

DES

IRA

BLE

PSY

CH

ROTR

OPH

ICG

RAM

-NEG

ATI

VE

BAC

TERI

A

Indi

cato

rstr

ains

Stra

ins

Pseu

dom

onas

fluor

esce

nsPs

eudo

mon

aspu

tida

Aer

omon

ashy

drop

hila

Phot

obac

teriu

mda

mse

lae

Asp

ergi

llus

flavu

sEs

cher

ichi

aco

liLi

ster

iam

onoc

ytog

enes

Salm

onel

laty

phim

uriu

mSt

aphy

loco

ccus

aure

usPs

eudo

mon

asae

rugi

nosa

Add

ition

Lact

ococ

cus

lact

isLc

1++

++++

+++

+++

++++

++++

++++

++++

+29

+Lc

2++

+++

+++

+++

++++

++++

+++

+++

25+

Lc3

+++

+++

+++

+++

+++

++++

+++

+++

++

26+

Lc4

++++

+++

+++

+++

+++

++++

+++

+++

26+

Lc5

+++

++++

+++

++++

++++

+++

+++

+25

+Lc

6++

+++

++++

+++

++++

++++

++++

-+

25+

Lc7

+++

+++

+++

++

+++

+++

++++

++

23+

Lc8

+++

+++

+++

++

+++

++++

+++

+22

+C

arno

bact

eriu

mpi

scic

ola

C1

++++

+++

+++

-+

++++

++++

++++

+++

+28

+C

2++

+++

+++

+++

+++

++++

++++

+++

+26

+C

3++

+++

++++

-+

++++

++++

+++

+++

+25

+C

4++

+++

+++

++++

+++

+++

++

20+

Leuc

onos

toc

mes

ente

roid

esLm

1++

+++

+++

+++

+++

++++

++++

++++

+++

29+

Lm2

++++

++++

+++

+++

+++

+++

+++

+++

+++

29+

Lm3

++++

++++

++++

+++

++++

++++

++++

+++

30+

Lm4

+++

++++

++++

+++

++++

+++

+++

++++

+29

+Lm

5++

+++

++++

+++

++++

++++

++++

+++

+28

+Lm

6++

+++

++++

++++

+++

++++

++++

++-

+27

+Lm

7++

+++

++++

+++

++++

++++

++++

++

26+

Lm8

++++

++++

+++

++

++++

+++

+++

+++

+27

+Lm

9++

++++

++++

+++

++++

+++

+++

++

26+

Lm10

+++

++++

++++

+++

+++

+++

++++

++

26+

Lm11

++++

+++

+++

+++

++++

+++

+++

++

25+

Lm12

++++

++++

+++

+++

+++

++++

++++

+26

+Lm

13++

++++

+++

++

+++

++++

++++

+++

+26

+La

ctob

acill

uspl

anta

rum

Lp1

++++

++++

++++

+++

+++

++++

++++

+++

+30

+Lp

2+

++++

+++

++

++++

+++

++++

+++

+25

+Lp

3++

+++

++++

+++

+++

+++

++++

+++

+26

+Lp

4+

+++

+++

++

+++

+++

++++

+++

22+

Lact

obac

illus

para

case

iLp

c1++

+++

+++

+++

++++

++++

+++

++++

+27

+Lp

c2++

+++

++++

++

++++

++++

+++

+++

+26

+Lp

c3++

+++

+++

+++

+++

++++

+++

+++

+25

+Lp

c4++

+++

++++

++

++++

+++

++++

-+

23+

Lact

obac

illus

brev

isLb

1++

++++

+++

+++

++

+++

++++

++++

+++

+29

+Lb

2++

++++

+++

++++

+++

+++

+++

++++

+++

29+

Lb3

++++

++++

+++

+++

++++

+++

+++

+25

+Lb

4++

+++

++++

+++

+++

+++

+++

+++

++

26+

Lb5

+++

++++

++

+++

+++

++++

++

21+

Lb6

++++

++++

+++

+++

++++

+++

21+

-:ne

gativ

ere

actio

n;+:

area

ofin

hibi

tion

zone

<20

0m

m2 ;

++:a

rea

ofin

hibi

tion

zone

betw

een

200

and

400

mm

2 ;++

+:ar

eaof

inhi

bitio

nzo

nebe

twee

n40

0an

d60

0m

m2 ;

++++

:are

aof

inhi

bitio

nzo

ne>

600

mm

2 .A

rea

ofha

loes

=P

(R2

-r2 )

;R,r

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the

halo

;r,r

adiu

sof

the

colo

ny.

Are

asof

inhi

bitio

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near

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ean

oftw

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ates

.



Table 4. No strain was totally susceptible to all antibiotics andmultiple resistances to most antibiotics were observed. Infact, all strains resisted to penicillin, norfloxacin, gentamicin,colistin, ciprofloxacin, nalidixic acid, erythromycin, tetracy-

clin and streptomicin. The majority of strains were suscep-tible to ampicillin, rifampicin and nitrofurantoin. However,a high percentage of isolates showed resistance to amikacin,ceftriaxome, chloramphenicol and cefuroxin.

TABLE 2. PROTEOLYTIC AND LIPOLYTICACTIVITIES OF 39 SELECTED STRAINS OFPSYCHROTROPHIC LACTIC ACID BACTERIA

StrainsAgar plate method(milk agar) Azocasein method

Lipolytic activity(fish fat)

Lactococcus lactisLc1 - 0.122 � 0.05 1.76 � 0.07Lc2 ++ 0.101 � 0.02 2.12 � 0.14Lc3 ++ 0.114 � 0.06 2.12 � 0.14Lc4 + 0.106 � 0.02 4.94 � 0.14Lc5 ++ 0.083 � 0.007 5.29 � 0.07Lc6 +++ 0.302 � 0.00 4.23 � 0.16Lc7 + 0.100 � 0.02 2.12 � 0.01Lc8 + 0.051 � 0.01 5.64 � 0.08Carnobacterium piscicolaC1 + 0.100 � 0.008 1.76 � 0.07C2 ++ 0.085 � 0.013 3.17 � 0.07C3 ++ 0.170 � 0.13 1.41 � 0.00C4 ++ 0.067 � 0.02 4.23 � 0.00Leuconostoc mesenteroidesLm1 + 0.089 � 0.02 1.76 � 0.07Lm2 + 0.165 � 0.11 2.47 � 0.07Lm3 +++ 0.094 � 0.002 4.23 � 0.14Lm4 + 0.102 � 0.016 1.76 � 0.07Lm5 + 0.089 � 0.03 1.76 � 0.07Lm6 + 0.088 � 0.00 5.29 � 0.21Lm7 + 0.130 � 0.018 4.23 � 0.00Lm8 + 0.120 � 0.01 3.53 � 0.12Lm9 + 0.072 � 0.00 2.47 � 0.03Lm10 + 0.097 � 0.05 1.76 � 0.02Lm11 + 0.095 � 0.10 4.23 � 0.06Lm12 - 0.062 � 0.002 4.23 � 0.16LM13 + 0.111 � 0.05 5.29 � 0.09Lactobacillus plantarumLp1 + 0.855 � 0.09 3.53 � 0.00Lp2 + 0.486 � 0.57 9.52 � 0.49Lp3 + 0.079 � 0.016 2.12 � 0.14Lp4 +++ 0.350 � 0.09 7.05 � 0.14Lactobacillus paracaseiLpc1 + 0.078 � 0.009 2.12 � 0.14Lpc2 +++ 0.085 � 0.007 4.94 � 0.09Lpc3 ++ 0.486 � 0.52 2.47 � 0.07Lpc4 ++ 0.167 � 0.09 2.82 � 0.14Lactobacillus brevisLb1 + 0.095 � 0.007 8.46 � 0.14Lb2 + 0.120 � 0.05 4.94 � 0.28Lb3 + 0.074 � 0.005 2.82 � 0.14Lb4 - 0.092 � 0.009 2.82 � 0.14Lb5 + 0.082 � 0.013 2.12 � 0.00Lb6 + 0.160 � 0.09 2.47 � 0.05

-: negative reaction; +: area of haloes surrounding the wells < 100 mm2; ++: area of haloessurrounding the wells between 100 and 200 mm2; +++: area of haloes surrounding thewells > 200 mm2.Areas of haloes surrounding the wells are the mean of two replicates.



TABLE 3. ENZYMATIC ACTIVITY DETECTEDUSING API-ZYM SYSTEM OF 39 SELECTEDSTRAINS OF PSYCHROTROPHIC LACTIC ACIDBACTERIA

Strains

Enzymes tested

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Lactococcus lactisLc1 0 5 0 0 40 40 10 0 0 10 5 5 40 0 10 30 0 0 0Lc2 0 5 5 5 40 30 10 5 5 10 10 5 20 0 10 40 30 0 0Lc3 0 0 0 0 30 30 20 0 0 5 20 0 30 0 5 20 5 0 0Lc4 0 5 5 5 40 30 10 0 0 10 20 5 40 0 10 30 10 0 0Lc5 0 0 5 5 30 40 10 0 0 20 10 0 30 0 5 10 10 0 0Lc6 0 5 5 5 40 30 20 0 0 10 30 5 40 5 10 40 10 0 0Lc7 5 5 5 5 40 40 10 0 5 5 10 5 40 0 5 5 5 5 5Lc8 0 0 0 0 40 30 10 0 5 10 5 0 40 0 5 20 5 0 5Carnobacterium piscicolaC1 0 5 10 0 40 30 20 0 5 10 10 0 40 0 10 20 0 0 0C2 0 5 10 5 40 30 20 0 0 10 10 0 10 0 20 30 20 0 0C3 0 5 5 5 40 30 10 0 0 10 20 5 40 0 20 30 10 0 0C4 0 5 5 5 40 30 10 0 0 10 10 0 40 0 0 0 5 0 0Leuconostoc mesenteroidesLm1 0 5 0 0 10 0 5 0 0 5 5 10 30 0 30 40 0 0 0Lm2 0 5 5 5 40 30 10 0 0 0 5 5 30 0 10 40 20 0 0Lm3 0 5 5 0 30 30 10 0 0 5 20 5 40 0 20 30 5 0 0Lm4 0 5 5 5 40 30 20 0 0 5 10 10 40 0 20 40 10 0 0Lm5 0 5 5 5 40 30 20 0 0 5 10 5 30 0 10 40 40 0 0Lm6 0 0 0 0 30 10 5 0 0 5 5 5 30 0 10 40 40 0 0Lm7 0 5 5 5 40 30 10 0 0 10 10 10 20 5 20 40 10 0 0Lm8 0 0 0 0 10 0 5 0 0 5 5 10 10 0 20 40 0 0 0Lm9 0 5 5 5 40 40 20 0 0 5 5 5 10 0 5 40 10 0 0Lm10 0 5 5 5 40 30 10 0 0 5 5 5 5 0 5 30 5 0 0Lm11 5 5 0 0 40 30 5 0 0 20 10 5 30 0 30 40 5 0 0Lm12 0 5 5 5 40 30 10 0 0 10 10 10 40 0 40 30 0 0 0Lm13 0 5 5 5 40 40 10 0 0 10 10 5 30 0 10 40 5 0 0Lactobacillus plantarumLp1 5 5 5 0 40 30 10 0 0 5 10 0 5 0 20 40 20 0 0Lp2 0 5 5 5 40 30 20 0 0 5 20 0 40 0 0 20 10 0 0Lp3 0 5 10 5 40 30 10 0 0 10 5 10 0 5 0 5 5 0 0Lp4 0 5 5 5 40 30 10 0 0 5 10 0 5 0 5 10 10 0 0Lactobacillus paracaseiLpc1 0 5 5 0 40 30 10 0 0 5 10 0 30 0 10 20 10 0 0Lpc2 0 5 5 5 20 30 10 0 0 5 5 0 40 0 5 10 5 0 0Lpc3 0 5 5 0 40 10 20 0 0 20 5 0 5 0 5 10 5 0 0Lpc4 0 10 10 10 30 40 20 0 0 10 10 0 40 5 10 40 10 0 0Lactobacillus brevisLb1 0 0 0 0 40 30 10 0 0 5 5 5 20 0 10 40 0 0 0Lb2 0 5 5 5 40 30 10 0 0 5 5 5 10 5 5 30 10 0 0Lb3 0 5 5 5 40 40 20 0 0 20 30 0 40 0 0 5 0 0 0Lb4 0 0 0 0 40 40 10 0 0 10 10 5 40 0 20 40 5 0 0Lb5 0 5 5 5 40 40 30 0 0 10 10 10 40 0 20 40 5 0 0Lb6 0 0 5 0 30 5 5 0 0 20 10 0 0 0 0 5 0 0 0

1: alkaline phosphate; 2: esterase (C4); 3: esterase lipase (C8); 4: lipase (C14); 5: leucine arylamidase;6: valine arylamidase; 7: cystine arylamidase; 8: trypsin; 9: a-chymotrypsin; 10: acid phophatase; 11:naphthol-As-Bi-phophohydrolase; 12: a-galactosidase; 13: b-galactosidase; 14: b-glucuronidase;15: a-glucosidase; 16: b-glucosidase; 17: N-acetyl-b-glucosaminidase; 18: a-mannosidase; 19:a-fucosidase.Enzymatic activity (approximate values) expressed as free nanomole hydrolyzed substrate wasobtained from the colour intensity. 0: no activity; 1: liberation of 5 nmol; 2: 10 nmol; 3: 20 nmol;4: 30 nmol; and 5: �40 nmol.



Acidifying Activity. After 72 h, the pH of LAB strainsdecreased to values lower than 4.38. In fact, the strains ofLc. lactis (Lc1), Lb. plantarum (Lp1), Lb. brevis (Lb1 and Lb2)and C. piscicola (C2) were those which showed the highestacidifying capacity (data not shown).

Growth Rate at Different Temperatures, SaltConcentrations and pH. The growth rate at differenttemperatures, pH and the tolerance of salt concentrations arelimiting factors affecting the persistence and competitivenessof the starter cultures. The best growth of LAB strains wasobtained at 37C in the presence of 0% NaCl and at pH 4.6.The growth rate at the different other trials was lower (datanot shown).

Identification by Molecular and Sequencing Analy-ses. On the basis of biochemical and technological proper-ties, 27 potential starter strains were selected. To ensure theirdefinitive identification, the selected isolates possessing themost important inhibitory effect and the lower proteolyticand lipolytic activities were identified using PCR amplifica-tion according to Scarpellini et al. (2002) and Zeppa et al.(2004). Therefore, 12 representative bacteria from the 27 iso-lates (two strains from each species) were chosen for sequenc-ing study. In fact, using 16S rRNA genes and the two primersFd1 and Rd1, only one band at almost 1500 bp was observedfor all strains (Fig. 1). Therefore, after sequencing and com-parison with the GenBank database, we founded that selectedstrains were similar to the strains Lb. plantarum ATCC 14917,Lb. brevis ATCC 27305, Lc. Lactis KF147, C. piscicola AT71101238000999 and L. mesenteroides ATCC 8293, as theyshowed 99% similarity to the sequence of the 16S rRNA geneof these species. Analysis of the sequences of the two strains ofLpc1 and Lpc2 displayed indeed 100% identity with the 16SrRNA gene of Lb. paracasei ATCC 25302.

DISCUSSION

Technological Properties of LAB Strains

All strains were further studied for characterisation of theantibacterial compounds and technological properties’studies. The majority of strains inhibited undesirable andspoilage psychrotrophic Gram-negative bacteria, due to alter-native or simultaneous acid, hydrogen peroxide and bacterio-cins inhibition (Franzetti et al. 2003). LAB were shown toinhibit the growth of Gram-positive bacteria because theirmembrane was thinner than those of Gram-negative, whichcontain many peptidoglucanes that may protect the cyto-plasmic membrane from the action of the antimicrobialcompound (Tantillo et al. 2002; Ammor et al. 2006). Further-more, we observed that LAB inhibited also Gram-negativebacteria. This result was asserted by Ben Moussa et al. (2008)

TABLE 4. ANTIBIOGRAM DETERMINED BY ANTIBIOTIC DISCS OF 39SELECTED STRAINS OF PSYCHROTROPHIC LACTIC ACID BACTERIA

Strains

Antibiotics

F/M RA AM C AN CXM CRO Addition MAR

Lactococcus lactisLc1 S M R R R R R 14R 0.83Lc2 M M R R R R M 13R 0.75Lc3 M S R R R R R 14R 0.83Lc4 R M R R R M M 13R 0.75Lc5 R M M R R R M 13R 0.75Lc6 S R R R R R R 15R 0.92Lc7 M R R R R R S 14R 0.83Lc8 S R R R R R R 15R 0.92Carnobacterium piscicolaC1 R S R R R R R 15R 0.92C2 M S R R R R M 13R 0.75C3 S S R R R R S 13R 0.75C4 S R R R R R S 14R 0.83Leuconostoc mesenteroidesLm1 S M R R R R R 14R 0.83Lm2 R R R R R R S 15R 0.92Lm3 M M R R R R S 13R 0.75Lm4 R S R R R R S 14R 0.83Lm5 R M R R R R M 14R 0.83Lm6 M M R R R R S 13R 0.75Lm7 R S S S M M S 10R 0.50Lm8 R R R R R R M 15R 0.92Lm9 R S R R R R M 14R 0.83Lm10 R R S R R R M 14R 0.83Lm11 R S R R R R M 14R 0.83Lm12 M M R R R R R 14R 0.83Lm13 S R R R R R S 14R 0.83Lactobacillus plantarumLp1 M S M R R R S 12R 0.67Lp2 M S R R R R S 13R 0.75Lp3 S R S R S S S 11R 0.58Lp4 S M R R R R R 14R 0.83Lactobacillus paracaseiLpc1 R M R R R R R 15R 0.92Lpc2 R R R R R M M 14R 0.83Lpc3 R R R R R M M 14R 0.83Lpc4 M R R R R R M 14R 0.83Lactobacillus brevisLb1 R R S R R R S 14R 0.83Lb2 R R S R R R S 14R 0.83Lb3 M R M R R R S 14R 0.83Lb4 M S S S S S S 8R 0.33Lb5 R R S R R R S 14R 0.83Lb6 M R M R R R S 14R 0.83Lb7 S R S R S S S 11R 0.58

All the strains were resistant to penicillin G (10 UI), gentamicin (500 mg),colistin (50 mg), ciprofloxacin (5 mg), norfloxacin (5 mg), nalidixic acid(30 mg), erythromycin (15 mg), tetracycline (30 mg) and streptomycin(500 mg).AN: amikacin (30 mg), AM: ampicillin (10 mg), CXM: cefuroxin (30 mg),CRO: ceftriaxom (30 mg), 5: F/M nitrofurantoin (300 mg), C: chloram-phenicol (30 mg), RA: rifampicin (30 mg).Antibiotic susceptibility was tested two times.R, resistant; S, sensitive; M, moderate; MAR, multiple antibioticresistance.



and Leroi (2009). Moreover, our results were in agreementwith those of Thapa et al. (2006) and Aymerich et al. (2008),who founded that species of Lactococcus showed antagonisticactivities against Listeria innocua, L. monocytogenes, Salmo-nella and S. aureus and also against P. aeruginosa, which canreduce the number of the undesired microorganisms in thefish products and preserve his quality (Einarsson and Lauzon1995; Aymerich et al. 2005). Proteolytic activity evaluatedusing azocasein methods revealed proteolytic capacity for allselected LAB strains. However, LAB do not have high pro-teolytic capacity compared with other group of bacteria. Infact, their activity is usually detected using specific substrateand/or sensitive tests. In our research, all LAB strains couldhydrolyze only casein and they showed weak proteolyticactivities in the fish products; these results were in agreementwith those reported by Thapa et al. (2006). Moreover, Monnetand Gripon (1994) observed that Lactobacilli are more pro-teolytic than Lactococci, which was in line with our findings.Results of lipolytic activity showed that all strains do not havelipolytic activity on agar medium and a poor activity whenusing fish fat. This confirmed the results reported by Ammoret al. (2005) and Ben Moussa et al. (2008), which foundedthat LAB strains are weakly lipolytic. The use of the API-ZYMmethod was of relevance for selection of strains as potentialstarter cultures on the basis of superior enzyme profiles, espe-cially peptidases and esterase, for accelerated maturation andflavour development of fish products. The absence of pro-teinases (trypsin and chymotrypsin) and the high peptidase(leucine arylamidase, valine and cystine arylamidase) andesterase-lipase (C4 and C8) activities produced by the pre-dominant LAB isolated from fresh fish were similar to thosereported by Thapa et al. (2004) and Thapa et al. (2006), whichhad potential technological implications by increasing desir-able flavour in the products. In our study, the antibiotic resis-tance of the LAB strains corroborated with those of Liasi et al.(2009) who found that LAB strains isolated from fermentedfish product were resistant to a large number of aminoglyco-sides, which include amikacin, streptomycin and gentamycin.

Furthermore, these isolates developed resistance to the lifesaving drugs such as tetracyclines (tetracycline) and quinolo-nes (nalidixic acid and norfloxacin) and also resisted to colis-tin. However, our results partially confirmed those reportedby Liasi et al. (2009) who found that all isolates were sensitiveto b-lactam group of antibiotic which include penicillin Gand ampicillin and were also susceptible to erythromycin,chloramphenicol and nitrofurantion. As evident in thepresent study, all the strains were multiresistant and showedmultiple resistance index (MAR) ranging from 0.33 to 0.92.The occurrence of MAR among the bacterial species could bea problem associated with transfer of resistance to otherorganisms of human/veterinary significance (Snoussi et al.2010). Furthermore, the most important characteristic forpotential starter strains was their ability to acidify their envi-ronment rapidly. In fact, the acid production and the accom-panying pH decrease give a specific aroma and extend the logphase of sensitive organisms including foodborne pathogens(Kostinek et al. 2007; Galvez et al. 2010). The combination oflow pH and organic acids (mainly lactic acid) is the mainpreservation factor in fermented fish products. Generally, pHshould be below 5–4.5 in order to inhibit pathogenic andspoilage bacteria (Owens and Mendoza 1985; Paludan-Muller et al. 2002). Besides, the ability of the starter culture tocompete with the natural microbiota of the raw material andto undertake the metabolic activities expected was condi-tioned by its growth rate and survival in the conditions pre-vailing in the fish (high salt concentrations, low temperaturesand low pH). None of the LAB isolates obtained from thesamples were halotolerant (i.e., 18% salt tolerance), whichwas in agreement with the result reported by Thapa et al.(2006) on fish products.

Molecular Identification and Sequencing

On the basis of DNA amplification and sequencing analyses,we founded that using 16S/23S Intergenic Spacer, all Lactoba-cillus strains presented two spacer region fragments of

FIG. 1. ELECTROPHORESIS OF PCR – AMPLIFIED 16S SPACER REGION OF SELECTED STRAINS OF PSYCHROTROPHIC LACTIC ACID BACTERIAISOLATED FROM FRESH FISHLane M, DNA molecular mass marker (100-bp ladder); Lp: Lactobacillus plantarum; Lc: Lactococcus lactis; Lpc: Lactobacillus paracasei;C: Carnobacterium piscicola; Lb: Lactobacillus brevis; Lm: Leuconostoc mesenteroides. PCR, polymerase chain reaction.



approximately 300 and 500 bp, which correspond to the twospacer regions of Lactobacillus genus named small and large.The larger one displayed differences in sequence betweenspecies (Berthier and Ehrlich 1998; Rachman et al. 2004).Also, strains of Leuconostoc and Lactococcus showed onespacer region at almost 300 bp; it corresponds to small frag-ment of Lactobacillus (Kabadjova et al. 2002; Ben Moussaet al. 2008). Moreover, using 16S rRNA genes, only one bandat almost 1500 bp was observed for all strains (Winker andWoese 1991). Our results suggested that, based on phenotypiccharacters and the API system, the identification of the 12selected strains was confirmed by molecular identification.

CONCLUSION

The results of the current study indicated that selected LABstrains originating from fish could constitute biopreservativetools for fresh fish and fish products. In fact, on the basis ofbiochemical and technological properties, their addition tofish may improve good microbiological and physicochemicalproperties, safety and then the stability of the productby extending his shelf life during refrigerated storage. Inour research, most of the studied and selected LAB canbe included in fresh fish for their important antagonisticactivity against spoilage and pathogenic bacteria and weakproteolytic and lipolytic activities.

ACKNOWLEDGMENTS

Theauthorsacknowledgefinancial support for this studyfromthe Ministère de l’enseignement supérieure et de la recherchescientifique, Tunisia. This work was realized in the unity ofresearch: Science and Technology of Foods (Research UnityUR04AGR02) Ecole Supérieure des Industries Alimentairesde Tunis.

A special thank to Pr. Maaroufi Abderrazek: Biologiste inPasteur Institut, Tunis.

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CHARACTERISATION, IDENTIFICATION AND TECHNOLOGICAL PROPERTIES OF PSYCHOTROPHIC LACTIC ACID BACTERIA ORIGINATING FROM TUNISIAN FRESH FISH