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Protein fingerprinting as a complementary analysis toclassical phenotyping for the identification of lactic acid

bacteria from Tenerife cheeseGraciela Pérez, Evaristo Cardell, Victoria Zárate

To cite this version:Graciela Pérez, Evaristo Cardell, Victoria Zárate. Protein fingerprinting as a complementary analysisto classical phenotyping for the identification of lactic acid bacteria from Tenerife cheese. Le Lait,INRA Editions, 2000, 80 (6), pp.589-600. <10.1051/lait:2000146>. <hal-00895446>

Original article

Protein fingerprinting as a complementary analysisto classical phenotyping for the identificationof lactic acid bacteria from Tenerife cheese

Graciela PÉREZ, Evaristo CARDELL, Victoria ZÁRATE*

Departamento de Microbiología y Biología Celular, Universidad de La Laguna,La Laguna 38206, Tenerife, Spain

(Received 21 February 2000; accepted 5 June 2000)

Abstract — A total of 125 lactic acid bacteria (LAB) from the genera Lactococcus, LactobacillusandLeuconostocisolated from Tenerife cheese were identified by both classical phenotypic and pro-tein fingerprinting methods. Classical identification revealed the presence of 11 different speciesand subspecies of LAB. Lactobacilli and leuconostocs identification was easy to achieve with the API50 CH system. By contrast, the identification of lactococci was difficult due to the heterogeneous andatypical profiles displayed by our strains. Sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE) of cell-free extracts (protein fingerprinting) has proved to be an efficient identificationmethod for LAB from Tenerife cheese. It generated complex and stable patterns that are easy tointerpret and compare with those of the reference strains. SDS-PAGE analysis could discriminate wellbetween the 8 different species and subspecies of LAB. However, the assignment of LAB strains toone of the Leuconostoc mesenteroidessubspecies required, in addition to protein fingerprinting, theperformance of two biochemical tests. When results of both identification methods were compared,23% of Tenerife cheese LAB isolates turned out to have been misclassified by the classical technique.

lactic acid bacteria / cheese / identification / protein fingerprinting

Résumé — L’empreinte digitale des protéines comme analyse complémentaire aux méthodesphénotypiques classiques pour l’identification des bactéries lactiques du fromage de Tenerife.Cent-vingt-cinq bactéries lactiques (LAB) des genres Lactococcus, Lactobacillus etLeuconostoc, iso-lées à partir du fromage de Tenerife, ont été identifiées par les méthodes phénotypiques classiques etpar la méthode d’empreinte digitale des protéines. Par identification classique, 11 espèces et sous-espèces différentes des LAB ont été mises en évidence. Les lactobacilles et leuconostocs ont étéfacilement identifiés à l’aide du système API 50 CH. Par contre, l’identification des lactocoquess’est révélée difficile en raison des profils hétérogènes et atypiques des souches. L’électrophorèseen SDS polyacrylamide (SDS-PAGE) de l’extrait cellulaire (l’empreinte digitale des protéines) s’est

Lait 80 (2000) 589–600 589© INRA, EDP Sciences

* Correspondence and reprintsTel.: (34) 922 318515; fax: (34) 922 630095; e-mail: vzarate@ull.es

G. Pérez et al.

1. INTRODUCTION

Lactic acid bacteria (LAB) are of greateconomic importance for the dairy and otherfermented food industries. LAB are Gram-positive, catalase negative, non-spore form-ing and have a fermentative sugarmetabolism with lactic acid as a major endproduct. The LAB found in cheeses consistprimarily of Lactococcus, Leuconostoc, andhomofermentative and heterofermentativeLactobacillus species.

Classical phenotypic identification ofLAB in dairy products depends mainly onphysiological and biochemical criteria. Iden-tification at the species level however, istime consuming and often ambiguous.Moreover, distinguishing between groupsof LAB such as leuconostocs and gas form-ing heterofermentative lactobacilli orbetween subspecies such as Lactococcuslactis ssp. lactisand ssp. cremoris, is diffi-cult and there have been many misclassifi-cations [11, 23]. Due to this fact, muchresearch has been directed towards thedevelopment of new phenotypic methodsthat improve the identification of thesemicroorganisms [15].

Modern taxonomy methods applied toLAB are based on molecular typing methodsand include both phenotypic and genotypicanalysis. Among phenotypic methods used,protein fingerprinting by SDS-PAGE of cellfree extracts or of cell wall proteins hasrevealed itself as a useful tool in the identi-fication of species and subspecies of LAB

[10, 12, 27, 30]. With regard to genotypictechniques, plasmid profile patterns ascer-tained by agarose gel electrophoresis [3, 6],ribotyping and randomly amplified poly-morphic DNA (RAPD) [5, 9, 13] have beensuccessfully used to resolve the taxonomicstatus of LAB.

Tenerife cheese is a traditional farmhousevariety produced on the island of Tenerife(Spain) from raw goat’s milk with the actionof natural microflora, the identification ofwhich is the first step towards the prepara-tion of a starter culture for use in large-scalemanufacture. Since the identification of thisflora on the basis of classical phenothypiccharacteristics has proved to be ambiguous[29], we decided to use a molecular typingmethod to improve its identification.

The aim of this study was to evaluate theefficiency of protein fingerprinting whenused as a complementary analysis to classi-cal phenotyping for the identification of125 LAB (lactococci, lactobacilli and leu-conostocs) strains isolated from Tenerifecheese.

2. MATERIALS AND METHODS

2.1. Strains, mediaand cultivation conditions

One hundred and twenty-five strains oflactic acid bacteria (LAB) from the generaLactococcus, Lactobacillus and Leuconos-toc were used in the present study. Thestrains have been isolated from 4 different

590

montrée efficace pour l’identification des LAB du fromage de Tenerife, car elle a conduit à des pro-fils complexes et stables faciles à interpréter et à comparer à ceux des souches de référence. L’ana-lyse par SDS-PAGE a permis de distinguer clairement 8 espèces et sous-espèces différentes de LAB.Cependant, l’identification des sous-espèces de Leuconostoc mesenteroidesa nécessité la réalisationde 2 tests biochimiques complémentaires. Quand on compare les deux méthodes d’identification, onconstate que 23 % des LAB isolées du fromage de Tenerife sont incorrectement identifiées par la tech-nique classique.

bactérie lactique / fromage / identification / empreinte digitale des protéines

Identification of LAB from Tenerife cheese

sentative members of the species and sub-species of LAB that were phenotypicallyidentified from Tenerife cheese, as well asother LAB and enterococci usually isolatedfrom Spanish raw goat’s milk cheeses [7,26]. All strains were maintained as frozenstocks at –80 °C in Man-Rogosa-Sharpemedium [4] (MRS, Unipath, Basingstoke,UK) containing 20% (v/v) glycerol. Work-ing cultures were prepared from frozen stockcultures by two consecutive transfers inMRS broth at 30 °C.

Tenerife cheeses at different maturationtimes as described previously [29] andassigned to genus by means of the followingtests: microscopic appearance in Gramstained preparations, catalase activity, CO2production from glucose in MRS broth with-out citrate using inverted Durham tubes andhydrolysis of arginine determined with theNessler reagent [14]. Reference strains usedfor protein fingerprinting (Tab. I) wereobtained from the Colección Española deCultivos Tipo (CECT) and included repre-

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Table I. Reference strains used for identification of LAB isolates by protein fingerprinting.Tableau I. Souches de référence utilisées pour l’identification des LAB isolées par l’empreintedigitale des protéines.

Name CECT Type Strain numbernb. strain as received

Enterococcus avium 968 T ATCC 14025 Enterococcus faecalis 481 T ATCC 19433 Enterococcus faecium 410 T ATCC 19434 Enterococcus gallinarum 970 T ATCC 35038 Lactobacillus acidophilus 903 T ATCC 4356 Lactobacillus brevis 216 CCRC 14060 Lactobacillus casei 475 ATCC 393 Lactobacillus cellobiosus 562 T ATCC 11739 Lactobacillus curvatusssp. curvatus 904 T ATCC 15601 Lactobacillus fermentum 4 007 T ATCC 14931 Lactobacillus paracaseissp. paracasei 4 022 T ATCC 25302 Lactobacillus paracaseissp. paracasei 277 ATCC 25598 Lactobacillus plantarum 748 T ATCC 14917 Lactobacillus plantarum 220 ATCC 8014 Lactobacillus rhamnosus 278 T ATCC 7469 Lactococcus lactis ssp. cremoris 697 T ATCC 19257 Lactococcus lactis ssp.lactis 185 T ATCC 9936 Lactococcus lactis ssp.lactis 4 041 IFPL 361 Lactococcus raffinolactis 988 T ATCC 43920 Leuconostoc lactis 4 173 T ATCC 19256 Leuconostoc mesenteroidesssp. cremoris 872 T ATCC 19254 Leuconostoc mesenteroidesssp. dextranicum 912 T ATCC 19255 Leuconostoc mesenteroidesssp. mesenteroides 219 T ATCC 8293 Leuconostoc mesenteroidesssp. mesenteroides 4 046 IFPL 704

ATCC, American Type Culture Collection, Rockville, Maryland, USA; CECT, Colección Española de CultivosTipo, Universidad de Valencia, Valencia, Spain; CCRC, Culture Collection and Research Center, Taiwan; IFPL,Instituto del Frío, Ciencia y Tecnología de los Productos Lácteos, Madrid, Spain.

G. Pérez et al.

2.2. Identification of strainsby classical phenotypic criteria

Identification at the species level of Lac-tococcuswas performed according to thecriteria of Schleifer and Kilpper-Bälz [21],Schleifer et al. [22] and Schleifer [20]. TheAPI 50CH galleries with API 50 CHLMedium (Bio Merieux, Marcy-l’Étoile,France) were used to identify the leuconos-tocs and lactobacilli due to the metabolismof 49 carbohydrates. Identification resultswere obtained with the aid of the APILABPlus (API, Bio Merieux) identification soft-ware.

2.3. Identification of strainsby SDS-PAGE of cell-free extracts(protein fingerprinting)

Cells were grown in 5 mL of MRS brothat 30 °C for 18 h. 1 × 1010 cfu were harvestedby centrifugation at 13000 g for 10 min andwashed twice with 1 mL of 50 mmol⋅L–1

Tris-HCl, pH 7.0. 50 mg of glass beads(150-200 µm, Sigma, St.-Louis, MO, USA)were added to the pellet. After mechanicaldisruption of the cells by vortexing during1 min, 100 µL of SDS-PAGE sample treat-ment buffer (150 mmol⋅L–1 Tris-HCl,pH 6.8; 4% SDS; 20% glycerol, 10% β-mer-capthoethanol; 0.005% bromophenol blue)was added and the samples were boiled for10 min. Whole cells and cell fragments wereseparated by centrifuging twice at 13 000 gfor 10 min in order to obtain the cell freeextracts.

Cell free extracts (20 µL) were loaded intovertical slab gels (80 × 70 × 0.75 mm) usinga Mini Protean II electrophoresis cell (Bio-Rad, Richmond, CA, USA) and analysed bySDS-PAGE (10% acrylamide (w/v) in theresolving gel and 4% acrylamide (w/v) in thestacking gel) by the method of Laemmli [16].Molecular weight markers purchased fromSigma were: myosin (205 kg⋅mol–1), β-galac-tosidase (116 kg⋅mol–1), phosphorylase b(97.4 kg⋅mol–1), bovine albumin (66 kg⋅mol–1),

egg albumin (45 kg⋅mol–1) and carbonicanhydrase (29 kg⋅mol–1). Gels were run for1 h at 20 mA and stained for 30 min with0.1% (w/v) Coomassie blue R-250, 40%(v/v) methanol and 10% (v/v) acetic acid.Gels were destained with 40% (v/v)methanol and 10% (v/v) acetic acid and pho-tographed using a Mitsubishi CCD-400Evideo camera with P90 video printer andK65HM thermal paper.

3. RESULTS

3.1. Identification of lactic acidbacteria by classical methods

On the basis of classical phenotypic anal-ysis, 22 strains of lactic acid bacteria thatwere homofermentative, Gram-positive,catalase-negative cocci were considered lac-tococci (Tab. II). Within the three tests com-monly used to differentiate enterococci andlactococci (growth at 45 °C, at pH 9.6 and inthe presence of 6.5% NaCl; positive for ente-rococci and negative for lactococci), most ofthe lactococci isolates were able to grow in6.5% NaCl and some of them were evenable to grow at 45 °C or at pH 9.6. Strainswere considered to be lactococci when up totwo of these physiological tests were posi-tive and to the genus Enterococcuswhenthey were all positive. The identification tothe subspecies level of Lactococcus lactiswas done according to the inability of Lc.lactis ssp. cremoristo hydrolyse arginine,in contrast to Lc. lactisssp. lactis. AmongLc. lactis ssp. lactisone strain was assignedto biovar diacetylactisbecause of its abil-ity to produce acetoin from citrate by theVoges-Poskauer reaction. Finally, one strainthat produced acid from raffinose and sor-bitol and did not hydrolyse arginine wasidentified as Lactococcus raffinolactis.

Gram-positive, catalase-negative, het-erofermentative cocci that did not hydrol-yse arginine were considered leuconostocs,while Gram-positive, catalase-negativerods were considered lactobacilli. Table III

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Identification of LAB from Tenerife cheese

to this, 71.9% of strains were Ln. mesen-teroidesssp. mesenteroides, 15.6% wereLn. mesenteroides ssp. dextranicumand onestrain was identified as Ln. lactis.

Phenotypic characterisation of lactobacillirevealed a predominance of the species Lac-tobacillus plantarum(52.1%) and Lb. para-caseissp. paracasei(36.6%) and the pres-ence in smaller numbers of Lb. curvatus,Lb. brevis, andLb. pentosus.

summarises the identification of both generaat the species level. Since the API 50 CHsystem does not distinguish between Leu-conostoc mesenteroidesssp. mesenteroidesand Ln. mesenteroides ssp. dextranicum,thecriterion followed to differentiate them isthe ability of the first subspecies to produceacid from L-arabinose (one of the carbohy-drates included in the API 50 CH gallery) incontrast to the second one [20]. According

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Table II. Physiological and biochemical characteristics of lactococci isolated from Tenerife cheese.Tableau II. Caractéristiques physiologiques et biochimiques des lactocoques isolés à partir dufromage de Tenerife.

Number of isolates 13 1 7 1

Growth at 10 °C 13a 1 7 1 45 °C 0 0 1 0

Growth in pH 9.6 4 0 0 04% NaCl 13 1 7 1 6.5% NaCl 7 0 7 0 0.1% methylene blue 13 1 6 1 0.04% Ktellurite 8 1 0 0

Survives 60 °C.30 min–1 13 1 6 1 Arginine hydrolysis 13 1 0 0 Acetoin from citrate 0 1 5 1 β-haemolysis 0 0 0 0

Acid from L-Arabinose 2 0 2 1 Glucose 13 1 7 1 Lactose 13 1 7 1 Maltose 13 1 7 1 Mannitol 5 0 6 1 Melezitose 1 0 6 1 Melibiose 2 0 6 1 Raffinose 1 0 2 1 Ribose 13 1 7 1 Sorbitol 8 1 4 1

Litmus milk test RACb 8 RAC 1 RAC 6 RAC 1AC 5

Identification Lc. lactisssp. Lc. lactis ssp. lactis Lc. lactis ssp. Lc. raffinolactis lactis biovar diacetylactis cremoris

a Number of positive isolates in each test; b R = reduction, A = acidification, C = coagulation.a Nombre d’isolats positifs pour chaque test ; b R = réduction, A = acidification, C = coagulation.

G. Pérez et al.

3.2. Identification of lactic acid bacteriaby protein fingerprinting

The analysis of cell protein extracts ofthe 125 strains of Tenerife LAB cheese iso-lates by SDS-PAGE showed six clearly dif-ferent profiles. After visual comparison ofthese electrophoretic patterns with those ofthe reference strains, the following speciesand subspecies were identified: Lactobacil-lus plantarum, Lb. paracasei ssp. paracasei,Lb. curvatus, Lactococcus lactis ssp. lactis,Lc. lactis ssp. cremoris and Leuconostocmesenteroides.

Figures 1 and 2 show the protein finger-printing identification of 14 of our LAB iso-lates (TF strains). Protein fingerprints ofLactobacillus plantarumand Lb. paracaseissp. paracaseiisolates from Tenerife cheesewere identical to that of their respective typestrains (Fig. 1). On the other hand, Lacto-bacillus curvatusssp. curvatusisolates wereslightly different when compared to the typestrain since the latter exhibited two clearbands below 45 kg⋅mol–1 while the formerlacked the lowest one (Fig. 1a, lanes 6 and 7).

Protein profiles of our Lactococcus lac-tis ssp.lactisstrains were almost the same asthe one exhibited by the type strain, exceptthat our isolates showed an additional bandbetween 45 and 29 kg⋅mol–1 (Fig. 2a, lanes 2to 5). In addition, no significant differenceswere found between the protein profilesof our Lactococcus lactis ssp. cremorisstrains and the type strain, though the clearband between 97.4 and 66 kg.mol–1 wasmore intense in our isolates (Fig. 2a, lanes 6and 7).

Protein fingerprints of type strains of Leu-conostoc mesenteroidessubspecies werevery similar (Fig. 2b, lanes 3, 5 and 7), mak-ing the identification of our strains by visualcomparison very difficult. In this case, thefollowing biochemical criteria were takeninto consideration to finally assign the Ln.mesenteroidesto one of the three subspecies:Ln. mesenteroides ssp. cremoris, inabilityto produce dextran when grown on solidmedia containing 0.5% sucrose; Ln. mesen-teroides ssp. mesenteroides, productionof dextran and of acid from L-arabinose;and Ln. mesenteroides ssp. dextranicum,

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Table III. Lactobacilli and leuconostocs from Tenerife cheese identified by the API 50 CH system.Tableau III. Souches de lactobacilles et leuconostocs du fromage de Tenerife, identifiées par lesystème API 50 CH.

Isolates Number %

Lactobacilli 71 100 Lactobacillus plantarum 37 52.1 Lb. paracaseissp. paracasei 26 36.6 Lb. curvatus 2 2.8 Lb. brevis 1 1.4 Lb. pentosus 1 1.4 Unidentified 4 5.7

Leuconostocs 32 100 Leuconostoc mesenteroidesssp. mesenteroides 23 71.9 Ln. mesenteroides ssp. dextranicum 5 15.6 Ln. lactis 1 3.1 Unidentified 3 9.4

Identification of LAB from Tenerife cheese 595

Figure 1.SDS-PAGE of cell-free extracts of lac-tic acid bacteria. (a) Lane 1, molecular weightmarkers; lane 2, Lactobacillus plantarumTF318;lane 3, Lb. plantarumTF236; lane 4, Lb. plan-tarumTF845; lane 5, Lb. plantarum type strain;lane 6, Lb. curvatus ssp. curvatus TF176; lane 7,Lb. curvatusssp. curvatus type strain; lane 8,Lb. brevistype strain. (b) Lane 1, molecularweight markers; lane 2, Lb. paracaseissp. para-caseiTF672; lane 3, Lb. paracaseissp. paraca-seiTF648; lane 4, Lb. paracaseissp. paracaseiTF271; lane 5,Lb. paracaseissp. paracaseitypestrain; lane 6, Lb. fermentumtype strain; lane 7,Lb. caseitype strain; lane 8, Lb. acidophilustypestrain.Figure 1.SDS-PAGE de l’extrait cellulaire desbactéries lactiques. (a) 1, Marqueurs de massemoléculaire ; 2, Lactobacillus plantarumTF318 ;3, Lb. plantarumTF236 ; 4, Lb. plantarumTF845 ; 5, Lb. plantarum souche type ; 6,Lb. curvatus ssp. curvatus TF176 ; 7, Lb. cur-vatusssp. curvatus souche type ; 8, Lb. brevissouche type. (b) 1, Marqueurs de masse molé-culaire ; 2, Lb. paracaseissp. paracaseiTF672 ;3, Lb. paracaseissp. paracaseiTF648 ; 4, Lb.paracaseissp. paracaseiTF271 ; 5,Lb. para-caseissp. paracaseisouche type ; 6, Lb. fer-mentumsouche type ; 7, Lb. caseisouche type ;8, Lb. acidophilussouche type.

Figure 2.SDS-PAGE of cell-free extracts of lac-tic acid bacteria. (a) Lane 1, molecular weightmarkers; lane 2, Lactococcus lactisssp.lactisTF400; lane 3, Lc. lactis ssp.lactisTF53; lane 4,Lc. lactis ssp.lactis TF61; lane 5, Lc. lactis ssp.lactis type strain; lane 6, Lc. lactisssp. cremorisTF165; lane 7, Lc. lactisssp. cremoristype strain;lane 8, Lc. raffinolactistype strain. (b) Lane 1,molecular weight markers; lane 2, Leuconostocmesenteroides ssp.mesenteroidesTF756; lane 3,Ln. mesenteroides ssp.mesenteroidestype strain;lane 4, Ln. mesenteroides ssp.dextranicum TF275;lane 5, Ln. mesenteroides ssp.dextranicumtypestrain; lane 6, Ln. mesenteroides ssp.cremorisTF197; lane 7,Ln. mesenteroides ssp.cremoristype strain; lane 8, Ln. lactistype strain.Figure 2. SDS-PAGE de l’extrait cellulaire desbactéries lactiques. (a)1, Marqueurs de masse molé-culaire ; 2, Lactococcus lactisssp.lactis TF400 ;3, Lc. lactis ssp.lactisTF53 ; 4, Lc. lactis ssp.lac-tisTF61 ; 5, Lc. lactis ssp.lactissouche type ; 6, Lc.lactisssp. cremorisTF165 ; 7, Lc. lactisssp. cre-morissouche type ; 8, Lc. raffinolactissouche type.(b) 1, Marqueurs de masse moléculaire ; 2, Leuco-nostoc mesenteroides ssp.mesenteroidesTF756 ;3, Ln. mesenteroides ssp.mesenteroidessouchetype ; 4, Ln. mesenteroides ssp.dextranicum TF275 ;5, Ln. mesenteroides ssp.dextranicumsouche type ;6, Ln. mesenteroides ssp.cremorisTF197 ;7, Ln. mesenteroides ssp.cremorissouche type ;8, Ln. lactissouche type.

(a)(a)

(b)(b)

G. P

érez et al.596Table IV. Comparison analysis of the identification of LAB from Tenerife cheeses by classical and protein fingerprintig methods.

Tableau IV. Analyse comparative de l’identification des LAB du fromage de Tenerife par la méthode classique et l’empreinte digitale des protéines.

Nb. Name after classical phenotypic analysis Nb. Name after SDS-PAGE analysis % ofisolates isolates coincidence

14 Lactococcus lactisssp. lactis 13 Lactococcus lactisssp. lactis 92.81 Lactobacillus plantarum

7 Lactococcus lactisssp. cremoris 1 Lactococcus lactisssp. cremoris 14.25 Lactobacillus plantarum1 Lactobacillus paracasei ssp. paracasei

1 Lactococcus raffinolactis 1 Lactobacillus paracasei ssp. paracasei 0.037 Lactobacillus plantarum 29 Lactobacillus plantarum 78.4

6 Lactobacillus paracasei ssp. paracasei1 Lactobacillus curvatus ssp. curvatus 1 Lactococcus lactisssp. lactis

26 Lactobacillus paracasei ssp. paracasei 25 Lactobacillus paracasei ssp. paracasei 96.1 1 Lactobacillus curvatus ssp. curvatus

2 Lactobacillus curvatus 1 Lactobacillus curvatus ssp. curvatus 50.01 Lactobacillus paracasei ssp. paracasei

1 Lactobacillus brevis 1 Lactobacillus curvatusssp. curvatus 0.01 Lactobacillus pentosus 1 Lactobacillus plantarum 0.04 Unidentified lactobacilli 4 Lactococcus lactisssp. lactis23 Leuconostoc mesenteroidesssp. mesenteroidesa 22 Leuconostoc mesenteroidesssp. mesenteroidesb 95.6

1 Leuconostoc mesenteroidesssp. cremorisb

5 Leuconostoc mesenteroidesssp. dextranicuma 5 Leuconostoc mesenteroidesssp. dextranicumb 1001 Leuconostoc lactis 1 Leuconostoc mesenteroidesssp. dextranicumb 0.03 Unidentified leuconostocs 2 Leuconostoc mesenteroidesssp. dextranicumb

1 Leuconostoc mesenteroidesssp. cremorisb

Tests performed for subspecies identification: production of: a acid from L-arabinose; b dextran and acid from L-arabinose.

Tests pour l’identification des sous-espèces : production de :a acide à partir du L-arabinose ; b dextrane et acide à partir du L-arabinose.

Identification of LAB from Tenerife cheese 597

production of dextran and inability to pro-duce acid from L-arabinose [20].

Tables IV and V show respectively thecomparison analysis of the identificationsof LAB from Tenerife cheese ascertainedby classical and protein fingerprinting meth-ods and the coincidence between both.

Lactococci were the LAB isolates thatgave the worse results in the comparativestudy since only 63.6% of the identifica-tions obtained by classical methods wereconfirmed by protein fingerprinting analysis(Tab. V). Among lactococci, only 14.2% ofthe classical phenotypically identified Lac-tococcus lactis ssp. cremorisremainedunchanged after SDS-PAGE analysis, whilethe rest of the strains turned out to be lacto-bacilli (Tab. IV). In addition, the Lc. raffi-nolactisstrain identified by the classicalmethod proved to be Lb. paracaseissp.paracaseiafter protein fingerprinting.

Fifty-five of sixty-one lactobacilli(77.5%) were correctly identified by API50 CH, as the identifications were confirmedby SDS-PAGE analysis. Apart from someclassical misidentifications within the gen-era, it is remarkable that the four presump-tive lactobacilli that gave an unacceptableprofile in the API system were finallyassigned to Lactococcus lactis ssp.lactisbyprotein fingerprinting analysis (Tab. IV).

With regard to leuconostocs, API 50 CHand protein fingerprinting identificationswere coincident in 84.4% of isolates. Thefive phenotypic misclassifications found byclassical methods were finally assigned toone of the three subspecies of Leuconostocmesenteroides after SDS-PAGE analysisfollowed by the determination of the pro-duction of dextran and of acid from L-ara-binose.

4. DISCUSSION

Classical identification of Tenerife cheeseLAB strains revealed the presence of 11 dif-ferent species and subspecies. The identifi-cation of leuconostoc and lactobacilli by theAPI 50CH system and the APILAB plusprogram was easy to perform. This tradi-tional standardized identification method isstill very useful and routinely used [17, 25]although it is relatively expensive, espe-cially when a great number of isolates are tobe identified, and it does not take intoaccount the recent progress in LAB taxon-omy [24]. In contrast, the identification ofspecies and subspecies of lactococci by clas-sical physiological and biochemical methodshas been difficult because of the heteroge-neous and doubtful profiles obtained, whichcomplicates the comparison with the

Table V. Coincidence of the identifications of LAB from Tenerife cheese by classical and protein fin-gerprinting methods.Tableau V. Coïncidence entre les identifications des LAB du fromage de Tenerife par la méthode clas-sique et l’empreinte digitale des protéines.

Group after classical Coincidence of classical and SDS-PAGEphenotypic analysis Nb. isolates analysis identifications

Nb. isolates %

Lactococci 22 14 63.6 Lactobacilli 71 55 77.5 Leuconostocs 32 27 84.4

Total 125 96 76.8

G. Pérez et al.

phenotypic characteristics reported in theliterature. Thus, our Lactococcus lactisssp.cremorisstrains showed a very atypical pro-file, growth at 6.5% NaCl, in 0.1% methy-lene blue and production of acetoin fromcitrate (Tab. II) when compared to the typestrain. Furthermore, this lactococci sub-species was present in an unexpectedly highproportion (7 out of 22 lactococci) consid-ering the difficulty of isolating the cremorisphenotype from natural sources [1]. Thesedisagreements, which were initiallyattributed to the different environmentalpressures of our isolates and the type strainsas has been previously reported [2, 8], werefinally considered to be a consequence ofthe misclassification of the strains.

Analysis of cell-free protein profiles per-formed as a complementary identificationmethod to classical phenotyping has beensuccessfully used in this study. Each LABspecies gave a complex and stable patternthat could be visually compared, for identi-fication purposes, with those displayed bythe reference strains. In strains such as Lac-tobacillus plantarumand Lb. paracasei ssp.paracasei, the protein pattern generated afterSDS-PAGE was identical to that of theirrespective type strains. The protein finger-prints of the rest of lactobacilli and lacto-cocci did not perfectly match with those oftheir type strains, though the differencesfound did not affect the identification byvisual comparison. These slight dissimilar-ities may be due to the different origin ofthe type strains and our strains, and theyhave also been noticed by other authorswhen identifying lactobacilli species iso-lated from naturally fermented Greek drysalami and cheese [19, 30].

Leuconostoc mesenteroidessubspeciesidentification by SDS-PAGE was very dif-ficult since the type strains displayed verysimilar protein patterns which complicatesthe visual comparison with our strains. Inthis case, two additional biochemical testshave to be performed in order to assign thesubspecies: dextran production from sucrose

which is negative for Leuconostoc mesen-teroidesssp. cremorisand positive for theother two subspecies, and acid productionfrom L-arabinose that is positive for Ln.mesenteroidesssp. mesenteroides and neg-ative for subspecies dextranicum. The dif-ficulty of the electrophoresis of cell freeextracts to distinguish between the Leu-conostoc mesenteroides subspecies has beenpreviously reported [27, 28], and it is prob-ably because they are very close phyloge-netic subspecies [18] that they give verysimilar protein patterns.

When the comparison was done betweenthe identification of Tenerife cheese LABby classical phenotypic and protein finger-printing methods, differences were foundamong the three genera under study. A goodcoincidence was found between the twoidentification methods in leuconostocs iso-lates (84.4%). The leuconostocs misclassi-fications obtained by the API system wereconfirmed to be Leuconostoc mesenteroidesafter SDS-PAGE and finally assigned to oneof the subspecies by determining the pro-duction of dextran and of acid from L-ara-binose. A coincidence of 77.5% was foundfor lactobacilli isolates. The majority of phe-notypically misclassified lactobacilli wereconfirmed to be different Lactobacillusspecies by protein fingerprinting, while onestrain of Lactobacillus plantarumas wellas the four presumptive lactobacilli thatcould not be identified by the API systemproved to be Lactococcus lactisssp. lactis(Tab. IV).

After protein fingerprinting, only 63.6%of classical phenotypically identified lacto-cocci were confirmed. It is remarkable thatonly one of seven isolates of Lactococcuslactis ssp. cremorisremained unchangedwhile the other six turned out to be Lacto-bacillus plantarumor Lb. paracaseissp.paracasei.

The fact that the misclassifications of lac-tococci were finally assigned to differentspecies of the genus lactobacilli and that

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five lactobacilli turned out to be lactococci,indicates that the major problem encoun-tered in the classical identification of LABwas the assignment to the genera Lacto-coccusor Lactobacilluswhich was basedon the microscopic observation of the strains(cocci cells were considered Lactococcuswhile rod shaped cells were considered Lac-tobacillus). The reason for this mistake isthat, under certain growth conditions, lac-tococci may form ovoid or even rod shapedcells and that lactobacilli may produce veryshort rods or ellipsoid cells [14] whichmakes the morphological determination dif-ficult. When the strains that were pheno-typically missclassified at the genus levelwere subject to new phenotypic identifica-tion, the results obtained were in agreementwith those obtained after SDS-PAGE anal-ysis (not shown).

The results of this work indicate that clas-sical biochemical and physiological testsare unsatisfactory for the identification ofLAB from Tenerife cheese, since 23.2% ofisolates were incorrectly identified follow-ing these methods. By contrast, the analysisof cell-free extracts provides an effectiveand reliable molecular-based typing methodthat generates complex and stable patternsthat are easy to interpret and compare. Whenprotein fingerprinting was performed as acomplementary analysis to classic pheno-typing the totality of the strains could becorrectly identified.

In conclusion, our results show that pro-tein fingerprinting analysis allows the reli-able differentiation of Tenerife cheese LABand can be applied to complement prelimi-nary identifications by classical phenotypictests.

ACKNOWLEDGMENTS

This work was financially supported by agrant from Cabildo Insular de Tenerife.

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