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LWT 40 (2007) 1146–1155

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Polyphasic characterization of indigenous lactobacilli and lactococcifrom PDO Canestrato Pugliese cheese

Lucia Aquilanti�, Emanuele Zannini, Annalisa Zocchetti,Andrea Osimani, Francesca Clementi

Department of Food Science, Polytechnic University of Marche, via Brecce Bianche, 60131 Ancona, Italy

Received 5 April 2006; received in revised form 4 September 2006; accepted 5 September 2006

Abstract

A polyphasic approach, involving both genotypic and phenotypic analyses was used to characterize 33 isolates of lactic acid bacteria

from the raw milk Protected Designation of Origin (PDO) Canestrato Pugliese cheese, in order to select candidate strains that can be

used as autochthonous starter cultures in the dairy industry. Randomly amplified polymorphic DNA (RAPD) analysis and clustering by

the unweighted pair-group method with arithmetic averages (UPGMA) were used to evaluate the genotypic diversity, while phenotypic

characterization was performed through miniaturized assays and traditional biochemical tests. Technological properties of major interest

for cheese-making (acidification, tendency to lysis, proteolytic and peptidase activity) were also evaluated, and selected subset of data was

statistically examined by the one-way ANOVA and the Tukey–Kramer HSD test, to highlight strain-specific and species-specific

differences among the isolates. A high degree of diversity appeared in the phenotypic and technological traits in opposition to a relatively

low genotypic diversity. Although none of the isolates showed the best performances in all the activities, an appropriate mixture of

strains could be selected for providing an efficacious autochthonous starter culture.

r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: PDO Canestrato Pugliese cheese; Autochthonous lactic acid bacteria; Starter cultures

1. Introduction

Canestrato Pugliese is a Protected Designation of Origin(PDO) cheese (CEE Regulation 1107/96) that is producedin Southern Italy from whole ewe’s milk. Nowadays,it is mainly manufactured with pasteurized milk that isinoculated with starter cultures, although in some dairyfarms it is still produced from raw milk without theaddition of starters. In the latter case, the ripening processdepends entirely on the indigenous microbial populationsthat contaminated the raw milk and the curd during themilking and cheese-making. The traditional technologythat is applied for the production of Canestrato Pugliesecheese has been previously described (Albenzio et al.,2001). According to its designation as a PDO cheese (CEERegulation 2081/92), the peculiar characteristics of Canes-

0 r 2006 Swiss Society of Food Science and Technology. Pu

t.2006.09.001

ing author. Tel.: +3971 2204985; fax: +39 71 2204858.

ess: l.aquilanti@univpm.it (L. Aquilanti).

trato Pugliese are strictly related to its production area,and particularly to the composition and diversity of theindigenous lactic acid bacteria (LAB) population, which ismainly responsible for the increase in the cheese flavourduring ripening (Steele & Unlu, 1992). The contribution ofthe indigenous LAB population to the biochemical andsensory characteristics of this PDO cheese, coupled to thehigh resistance to the selective conditions of the cheese-making (De Angelis et al., 2001) encourage the selection ofindigenous strains as new starter cultures for the dairyindustry. Accordingly, preservation of the genetic pool ofstrains that have developed in dairy environments that arenot yet affected by selected industrial cultures is of primaryimportance (Mannu & Paba, 2002). As selected startersconsist of either a single or a few well-characterized strains,the natural microbial population of raw milk cheeses isgenerally much more complex.To date, several genotypic and phenotypic methods have

been developed for the characterization of LAB strains

blished by Elsevier Ltd. All rights reserved.

ARTICLE IN PRESSL. Aquilanti et al. / LWT 40 (2007) 1146–1155 1147

from dairy products. Randomly amplified polymorphicDNA (RAPD) analysis is a PCR-based method that iswidely used for estimation of the genotypic diversityamong Lactobacillus and Lactococcus strains (Du Plessis& Dicks, 1995), and it provides good levels of discrimina-tion while being applicable to a large number of cultures.However, a comprehensive characterization of thesemicroorganisms cannot ignore the evaluation of some oftheir phenotypic and technological traits (Vandamme et al.,1996), like the fermentation pattern, the acidifying andpeptidase activities, the proteolysis and tendency to lysis.Evaluation of these last traits is particularly important forthe selection of new starter strains, which should be able totransform the principal components of the curd into moresimple substances, either as well-defined cellular entities orthrough the released enzymes.

In the present study, a polyphasic approach for thegenotypic and phenotypic characterization of 33 auto-chthonous LAB isolates from starter-free CanestratoPugliese cheese was developed. Molecular typing by RAPDanalysis, phenotypical and technological tests allowed aneffective strain differentiation to be achieved, enhancingthe sensitivity of each device used and leading to theindividuation of strains that will be potentially utilized asstarter cultures in the dairy industry.

2. Materials and methods

2.1. Reference strains

Nine strains from international culture collections wereused as references: Lactobacillus brevis DSM20054,DSM20556, DSM2647, DSM1267; Lactobacillus casei

DSM20011; Lactobacillus plantarum DSM20174,DSM2601; Lactococcus lactis ssp. cremoris DSM20069,DSM20388; Lb. casei NCIMB4114 and ATCC11578. Theywere purchased from (i) the ‘Deutsche Sammlung vonMikrorganismen und Zellkulturen’ (DSMZ, Braunsch-weig, Germany, http://www.dsmz.de/); (ii) the ‘AmericanType Culture Collection’ (ATCC, Manassas, USA, http://www.atcc.org/); (iii) the ‘National Collections of Industrialand Marine Bacteria’ (NCIMB, Aberdeen, Scotland,United Kingdom, www.ncimb.co.uk/).

2.2. Isolation and identification

The isolates were collected and identified as previouslydescribed by Aquilanti, Dell’Aquila, Zannini, Zocchetti,and Clementi (2006).

2.3. RAPD analysis

Bacterial cells were grown overnight at 30 1C in 5mlMRS broth (Oxoid, Basingstoke, UK). Bacterial DNA wasrecovered following the phenol–chloroform extractionmethod described by de Los Reyes-Gavillan, Limsowtin,Tailliez, Sechaud, and Accolas (1992). The amplifications

were performed in 25 ml reaction volume, containing10mM Tris–HCl (pH 8.3), 50mM KCl, 200 mM of eachdATP, dGTP, dCTP and dTTP, 3mM MgCl2, 1 mMprimer, 80 ng DNA and 2U Taq DNA polymerase(Finnzymes Oy, Keilaniemi, Finland). The PCR reactionswere carried out in a Gene Amp PCR System 9700 (PerkinElmerApplied Biosystem, Foster City, CA) using a 10-merprimer (50AGC AGC GTG G 30) (Cocconcelli, Porro,Galandini, & Senini, 1995), under the following conditions:initial denaturation at 94 1C for 2min, followed by 35cycles of denaturation at 94 1C for 1min, annealing at42 1C for 1min, elongation at 72 1C for 90 s, and a finalelongation step at 72 1C for 10min. The PCR productswere separated by electrophoresis on 1.5% (w/v) agarosegels in TBE (Tris–borate–EDTA [pH 8]) buffer. A 50-bpDNA molecular mass marker (Amersham Pharmacia,Uppsala, Sweden) was used as the size standard. TheDNA fragments were stained with ethidium bromide andviewed under UV light, at 254 nm. Electronic images of thegels were visualized by means of an ImageMaster VDS(Amersham Pharmacia), with capturing through theLISCAP 1.0 program (Amersham Pharmacia), and storedas TIFF files. The reproducibility of the RAPD profileswas tested by replicates in triplicate of all of the referencestrains. The band patterns were normalized and furtherprocessed with the GelCompar 4.0 software (AppliedMaths, Kortrijk, Belgium). Cluster analysis was performedby using the Dice similarity coefficient (Dice, 1945) and theunweighted pair-group method with arithmetic averages(UPGMA) (Sokal & Michener, 1958), with the 1.8 NTSYSsoftware (Rohlf, 1993). An 80% similarity was arbitrarilyselected as a threshold for the definition of the homo-geneous RAPD-based clusters.

2.4. Phenotypic characterization

The cultures were propagated in modified APTbroth (Difco Laboratories, Detroit, USA) with a16-h-incubation at 30 1C for lactococci and 37 1C forlactobacilli. The acid production from carbohydrates(cellobiose, galactose, maltose, mannitol, melibiose,ribose, sucrose, trehalose, arabinose, glycerol, fructose,glucose, lactose, mannose, melezitose, raffinose, rhamnose,D-xylose), the hydrolysis of esculin (esc), the tolerance to20, 40, 65 g/l NaCl, and the D- or L-lactic acid (D/lac, L/lac)production were evaluated by using a miniaturized assay inmicrotitre plates as previously described (Parente, Rota,Ricciardi, & Clementi, 1997). Arginine hydrolysis wastested in a modified MRS medium (MRS added with 3 g/lof arginine and sodium citrate in place of ammoniumcitrate). The isomer of lactic acid was determined by themethod of Von Krush and Lompe (1982) using 5-day-oldbroth cultures in modified MRS (MRS without meatextract, and added with 10 g/l of tryptone in place ofpeptone). Dextran production was tested as described bySchillinger and Lucke (1987).

ARTICLE IN PRESSL. Aquilanti et al. / LWT 40 (2007) 1146–11551148

2.5. Technological characterization

The acidifying and protease activities were assessed insterilized skim milk (SSM; pH 6.50) (Oxoid) at 37 1C forlactobacilli and 30 1C for lactococci, using standardizedinocula. The cultures were revitalized in MRS broth,harvested at 4000� g for 10min, washed twice with steriledistilled water, and re-suspended in 8.5 g/l NaCl (w/v).Aliquots of 0.5ml of the cell suspensions were inoculated intriplicate into 9.5ml of SSM; these gave optical densities of1.25 at 620 nm after a 1:10 dilution and they containedabout 109 cfu/ml. After 0, 8 and 24 h of incubation, 1-mlsamples were aseptically withdrawn for pH measurements,together with 1-ml aliquots of SSM as the controls, using a9224 model pH-meter (Hanna Instruments, Padova, Italy)that was equipped with an Eppendorf electrode (Hamilton,Reno Nevada, USA). Similarly, after 24 h and 7 days ofincubation, 1.5-ml aliquots were removed from each SSMculture and used for proteolysis assessment using theo-phthaldialdehyde (OPA) assay (Church, Swainsgood,Porter, & Catignani, 1983) after protein precipitation withtrichloroacetic acid (Oberg, Weimer, Moyes, Brown, &Richardson, 1991). The measurements were carried out at340 nm with an 8452 model Hewlett-Packard spectro-photometer (Waldbronn, Germany). The results werecalculated from a calibration curve obtained from dilutionof glycine in distilled water and were expressed as mg/mlglycine (Gly).

Cultures to be subjected to the assessment of thepeptidase activity were revitalized, centrifuged and washed,in duplicate, as described above. The final pellets werere-suspended in 1ml of 25mM 2-N-morpholino-ethanesul-phonic acid (MES) at pH 6.00. This suspension was pre-cooled to 4 1C (Williams, Felipe, & Banks, 1998) andmechanically disrupted by vortexing with glass-beads(150–212-mm diameter) (Sigma-Aldrich) for 30 cycles of30 s, with each cycle being followed by 30 s cooling. Thecrude cell-free extracts were finally separated by centrifu-gation at 8800� g at 4 1C for 1min, and then stored at�20 1C. The peptidase activities were determined using thefollowing synthetic substrates: L-Lys-, L-Glu-, L-Pro-,L-Gly-L-Pro-, L-Arg-L-Pro-p-nitroanilide (p-NA). The assaymixtures contained 195 ml of a 50mM potassium–pho-sphate buffer, pH 7.90, 90 ml of 0.5 g/l NaNO3 (v/v) and75 ml of the cell-free extract. Thirty ml of a 20mM solutionof each p-NA derivative was added separately to thismixture. After an incubation at 37 1C for 1 h, the reactionwas stopped by adding 0.9ml of an acqueous solution ofacetic acid (100ml/l). The samples were centrifuged at8800� g for 5min, and the absorbance was measuredat 410 nm. The concentrations of p-nitroanilide werecalculated using the molar absorption coefficient value(8800mol/l cm) (Rollan & Font de Valdez, 2001). One unit(U) of enzymatic activity corresponds to the amount ofenzyme that releases 1 mmol of p-nitroanilide/min under theassay conditions used. The specific enzyme activities areexpressed as U mg protein. The total amount of protein in

the cell-free extracts was quantified by the dye-bindingmethod of Bradford (1976). All of the synthetic substrateswere purchased from Sigma Chemical (Pool, UK) andBachem (Saffron Walden, UK). The kinetics of autolysiswere determined as described by Martınez-Cuesta, Pelaez,Juarez, and Requena (1997). Briefly, lactococci andlactobacilli were grown overnight in MRS broth at 30and 37 1C, respectively. Absorbance was measured at650 nm. One-millilitre aliquots of 0.7 O.D. cell suspensionswere centrifuged at 6000� g for 5min. The harvestedpellets were washed twice in 8.5% NaCl (w/v) and finallyre-suspended in 1ml lysis buffer (0.2M NaCl, adjustedto pH 5.5). The autolysis kinetics were monitored bymeasuring the O.D. of the cell suspensions at 650 nm,after 0 and 30min and after 8 h of incubation at40 1C. The results are expressed according to Langsrud,Landaas, & Castberg (1987), in terms of the autolysis ratesper h (AR) and the autolysis percentages (%), using thefollowing formulas, respectively: (AR) ¼ (A3–A2)/0.5;(%) ¼ 100–A1/A3� 100, where A1 is the final O.D., A2is the O.D measured after 30min, and A3 is the initial O.D.

2.6. Statistical analysis

Three repetitions of each phenotypic assay were per-formed. The arithmetic means and standard deviationswere calculated. A one-way analysis of variance (ANO-VA), along with the Tukey–Kramer honestly significantdifference (HSD) based on three replicates was carried outusing the Statistica software package (version 5.1, StatSoftInc., Tulsa, OK) with the following variables: VAR1,species, VAR2, strains within the same species; VAR3,autolysis rate, VAR4, acidifying activity at 8 h, VAR5,release of Gly at 24 h.

3. Results

3.1. RAPD and cluster analysis

Cluster analysis of the RAPD profiles from the 33 LABfrom starter-free Canestrato Pugliese cheese (Table 1), wereperformed separately within each species, since the absenceof a species-specific DNA band or a combination of bandsdid not allow a clear genotypic characterization when thestrains of the four species were all analysed together.As a whole, the 33 isolates were grouped into 14 different

genotypes that corresponded to single strains or clusters ofstrains. In more detail, it was possible to define: 2 clustersand 4 strains for Lb. plantarum (Fig. 1a); 3 clusters and 2independent strains for the species Lb. brevis (Fig. 1b); 1cluster and 1 strain for Lc. lactis subsp. cremoris (Fig. 1c);and 2 strains for Lb. casei (Fig. 1d).

3.2. Phenotypic characterization

Results of the 21 phenotypic tests are shown in Table 2.All of the isolates produced L-lactate either alone or

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Table 1

The indigenous lactic acid bacteria that were isolated from the raw milk,

curd and 1-, 2- and 4-week-old ripened Canestrato Pugliese cheese from

three dairy farms (F1, F2, F3) in the Apulia region (Southern Italy)

Species Isolate Farm Isolation source

Lactobacillus brevis 8 F1 Milk

14

45 1-week-old cheese

46

48

68 4-week-old cheese

77

81

83

86

49 F2 2-week-old cheese

58

72 F3 4-week-old cheese

73

78

Lactobacillus plantarum 29 F1 Curd

33

55 2-week-old cheese

23 F2 Curd

56 2-week-old cheese

59

17 F3 Milk

18

19

35 Curd

74 4-week-old cheese

Lactobacillus casei 30 F1 Curd

27 F3 Curd

Lactococcus lactis subsp. cremoris 4 F2 Milk

6 F3 Milk

28 Curd

51 1-week-old cheese

52 1-week-old cheese

L. Aquilanti et al. / LWT 40 (2007) 1146–1155 1149

together with D-lactate, and were resistant to 20 g/l NaCl.At the same time, except for two out of the five isolates thatbelonged to the species Lb. brevis and Lb. plantarum, theywere all able to ferment lactose and galactose, as well asglucose. None of the isolates either produced dextran orfermented D-arabinose.

3.3. Technological characterization

Further characterization of the isolates was carried outwith the aim of evaluating their potential for being used asindigenous starter cultures. As shown in Table 3 a widestrain-to-strain variability was observed with respect toboth the acidifying rate, assessed as the pH values after 8 h,and the acidifying power, assessed as the pH values after24 h. The majority of the isolates showed pH values lowerthan 6.00 within 8 h. Three cultures, namely isolates 56, 59and 73, that were characterized by both high acidifying

velocity and power revealed to be particularly suitable asstarters.The proteolytic activities, which were expressed as the

release of Gly after 24 h, were generally between 98.16 and184.71 mg/ml, except for a few isolates that were character-ized by notably higher (isolates 58 and 33) or lower(isolates 77 and 17) activities. These values did not increasemarkedly after 7 days of incubation, probably due to abalance between release and consumption of Gly that wasbeing progressively established during the assay. Anexception to this behaviour was seen for the isolates thatled to relevant increases in the final Gly levels (isolates 81,74 and 45) and those that, in contrast, led to notabledecreases (isolates 58 and 33).A high degree of diversity among the isolates was also

observed regarding their tendencies to lysis. Most of theisolates with the lowest percentages of lysis were ascribedto the species L. lactis subsp. cremoris, whereas the highestvalues were detected within the species Lb. brevis.Outcomes related to the amino-peptidase and dipeptidyl-

peptidase activities are shown in Table 4. The peptidaseactivities were among the highest found in this study, andthey were towards all of the substrates for isolates 58, 72and 18. Thus, isolate 72 showed the highest activity onL-Lys- and L-Gly-L-Pro-p-NA, whereas isolate 18 showedthe highest activity towards L-Glu- and L-Arg-L-Pro-p-NA.

3.4. Statistical analysis and biotypes definition

To determine if there were significant intra-species andinter-species differences among the isolates, statisticalanalyses by one-way ANOVA and Tukey–Kramer HSDwere carried out on the data sets for the acidifyingvelocities (pH value after 8 h), proteolytic activities (after24 h) and autolysis rates. Significant differences among thefour species under study were seen for the acidifying(Po0.001) and autolytic (P ¼ 0:040) activities, but not forproteolysis (P ¼ 0:749). Lb. brevis was revealed to bethe fastest acidifying species (mean, 5.78), followed byLb. plantarum (mean, 5.92), Lc. lactis subsp. cremoris

(mean, 6.03) and Lb. casei (mean, 6.25). For the autolysisrates, Lb. brevis and Lb. casei were characterized by meanvalues (0.07 and 0.06, respectively) that were markedlylower than those recorded for Lc. lactis subsp. cremoris andLb. plantarum (means, 0.13 and 0.14, respectively).The one-way ANOVA and Tukey–Kramer HSD also

allowed strain-dependent differences to be highlightedwithin each species, and 22 biotypes to be thus defined,each of which included isolates with similar proteolytic,acidifying and autolysis activities (Table 5). No correspon-dence between these biotypes and the RAPD-based clusterswere observed.

4. Discussion

Data of phenotypic characterization clearly demon-strated that these tests are not reliable enough for the

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0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

55

74

17

35

33

18

19

56

59

23

29

27

30

6

28

4

51

52

Lcl1

Lcl2

Lbc1

Lbc2

8

83

81

68

73

77

78

58

72

86

14

45

46

48

49

Lbb1

Lbb2

Lbb3

Lbb4

Lbb5

Lbp1

Lbp2

Lbp3

Lbp4

Lbp5

(d)

(c)

(b)

(a)

Fig. 1. The clustering of the 33 indigenous lactic acid bacteria isolated from the Canestrato Pugliese cheese on the basis of UPGMA-dendrograms from

RAPD profiles within the species Lb. plantarum (a), Lb. brevis (b), L. lactis subsp. cremoris (c), and Lb. casei (d). An 80% similarity was arbitrarily chosen

as a discriminating threshold to define the homogenous clusters, with each corresponding to a genotype.

L. Aquilanti et al. / LWT 40 (2007) 1146–11551150

identification of LAB at the species level. Indeed, as shownin Table 2, several isolates were characterized by atypicalmetabolic and biochemical behaviours respect to those

reported in the scientific literature for Lactobacillus

(Kandler & Weiss, 1984) and Lactococcus (Bottazzi,1993) species. One major reason for this mismatch might

ARTIC

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PRES

STable 2

The acid production by the 33 lactic acid bacteria isolated from the Canestrato Pugliese cheeses from 19 carbohydrates: cellobiose (cel), galactose (gal), lactose (lac), maltose (mal), mannitol (mnt),

melibiose (melb), raffinose (raf), ribose (rib), sucrose (suc), trehalose (tre), D-arabinose (D/ara), L-arabinose (L/ara), glycerol (gly), fructose (fru), glucose (glu), mannose (man), melezitose (melz),

rhamnose (rha), D-xylose (xyl)

RAPD cluster Isolate ara cel fru gal suc gly glu lac mal mnt man melz melb raf rha rib tre xil esc NaCl 20 g/l NaCl 40 g/l NaCl 65 g/l D/lac L/lac arg dex

Lbb1 8 + +a + + + � + + + + +a + + + +a + +a� + + + + + + � �

Lbb3 14 + +a + + � � + + + + � � + � � + +a + + + + + + + + �

45 �a� + + + � + + + � � � + � � + � + � + + + + + + �

46 + +a + + � � + + + + � � + � � + +a + + + + + + + + �

48 �a +a + + � � + + �

a� +a

� �a

� � + +a� + + + � + + � �

49 �a +a + + � � + + �

a� +a

� �a

� � + +a� + + + � � + + �

58 + +a + + + � + + + + +a� + � +a + +a + + + + + + + + �

68 + +a + + + + + + + + +a� + � +a + +a + + + + + + + + �

Lbb5 72 + � �a� � � + � + � � � �

a� � + � + � + + � + + + �

73 + � �a� � � + � + � � � �

a� � + � + � + + � + + + �

77 + +a + + + � + + + + +a� + � +a + +a + + + + + + + + �

78 + +a + + + � + + + + +a� + � +a + +a + + + + + + + + �

Lbb2 81 + +a + + + � + + + + +a + + + � + +a + + + + + + + + �

83 + � + + � � + + + � � � + + � �a +a

� + + + + + + + �

Lbb4 86 + � + � � � + � + � � � � � � + � + � + + � + + � �

Lbp3 18 + + + + + + + + + + + + + + +a + + + + + + + + + + �

19 + + + + + � + + + + + + + + +a + + + + + + + + + + �

33 + + + + + + + + + + + + + + +a + + + + + + + + + + �

35 + + + + + � + + + + + + + + +a + + + + + + + + + + �

56 + + + + + � + + + + + + + �a +a + + + + + + + + + + �

59 + + + + + � + + + + + � + �a� + + + + + + + + + + �

Lbp2 17 + �a + + + � + + + �

a�

a� + �

a +a + �a + + + + � + + � �

74 + �a + �

a�

a� + �

a + �a�

a� �

a�

a� + �

a + � + + � + + + �

Lbp4 23 � + + + �a� + + �

a�

a + � �a

�a� + + � + + + � � + + �

Lbp5 29 + �a�

a�

a�

a� + �

a + �a�

a� �

a�

a� �

a + � � + + � + + � �

Lbp1 55 + + + + + � + + + + + + + �a +a + + + + + + + + + + �

Lbc1 27 � + + + � � + + + + + � � � � + + � + + + � � + + �

Lbc2 30 � + + + � � + + + + + � � � � + + � + + + � � + + �

Lcl1 4 � + + + + � + + +a + + � � � � + +a +a +a + + � � + +a�

Lcl2 6 +a + + + +a� + + +a + + + +a +a

� + +a +a +a + +a +a +a + +a�

28 +a + + + � � + + +a + + + � � � + +a� +a + � � � + +a

�

51 � + + + +a + + + +a + + + +a� � + +a

� +a + +a� � + +a

�

52 � + + + +a� + + +a + + � � � � + +a

� +a + +a� � + +a

�

The hydrolysis of esculin and arginine, the tolerance to 20, 40 and 65 g/l NaCl, the production of L- or D-lactic acid and dextran from these lactic acid bacteria are also shown.

+ Positive reaction.

� Negative reaction.aAtypical behaviour respect to that reported in the scientific literature for Lactobacillus (Kandler & Weiss, 1984) and Lactococcus spp. (Bottazzi, 1993).

L.

Aq

uila

nti

eta

l./

LW

T4

0(

20

07

)1

14

6–

11

55

1151

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Table 3

The acidifying, proteolytic and autolytic activities of the lactic acid bacteria isolated from the Canestrato Pugliese cheese

Isolate Species Acidification Proteolysis Autolysis

8 h 24 h 24 h 7 days % AR

8 Lactobacillus brevis 6.3870.01 5.4770.01 135.2570.00 141.43730.60 32.1072.69 0.0370.01

14 5.9170.01 4.8070.01 147.62734.97 166.16756.83 39.3770.10 0.0370.01

45 5.6470.01 4.7170.01 147.62717.48 512.3874.37 20.7372.13 0.0670.01

46 5.7670.01 4.9870.00 141.4374.37 153.878.74 90.7771.75 0.0170.00

48 5.4970.01 4.8270.10 141.43713.11 141.43721.85 26.7571.03 0.1570.03

68 5.4870.04 4.4870.01 135.1074.37 150.8070.00 69.4870.73 0.1070.01

77 5.6370.05 4.8270.03 28.2974.37 37.5774.37 45.2072.13 0.1270.03

81 5.7670.00 4.8470.00 129.0570.10 673.40710.20 4.6071.46 0.0770.00

83 5.8670.00 5.1470.10 134.0772.14 153.0870.00 23.2573.93 0.0470.01

86 5.8670.01 5.1470.10 135.2570.00 150.2171.60 31.9571.91 0.0770.01

49 5.7370.02 4.9070.02 98.1674.37 104.3470.00 39.8473.06 0.0270.00

58 5.9170.00 4.7270.01 401.10721.85 184.71713.11 17.7571.38 0.0470.02

72 5.7670.00 4.8470.00 129.077 8.47 147.62730.60 49.0672.82 0.1470.01

73 5.3670.03 4.6070.05 135.2570.00 153.8070.00 38.7471.56 0.0870.03

78 6.1670.01 5.6770.00 122.8970.00 209.4474.37 26.2975.32 0.1470.08

29 Lactobacillus plantarum 5.9570.02 4.9370.22 184.7174.37 209.44748.08 19.7479.20 0.1370.06

33 5.8870.01 4.8870.01 376.37721.85 184.71739.34 24.7674.50 0.0870.00

55 6.1470.01 5.8370.02 116.7070.00 104.3570.00 28.5973.21 0.2270.04

23 6.1870.00 5.3170.01 122.8974.37 98.1674.37 9.2873.44 0.0170.00

56 5.5170.01 4.4170.10 116.8070.00 104.3570.14 24.5875.38 0.0770.03

59 5.5170.01 4.4170.01 135.25721.85 116.70743.71 13.4072.96 0.6070.02

17 6.3870.01 5.4770.01 29.5370.00 51.7970.00 27.0672.04 0.0770.02

18 5.5370.01 4.7670.01 184.7174.37 209.46721.85 17.1173.20 0.0770.02

19 6.2870.01 5.4070.00 135.2570.00 141.43730.60 19.2774.82 0.0570.03

35 6.2970.01 5.7070.01 166.16717.00 104.3474.30 7.0673.25 0.0570.02

74 5.5670.00 4.7970.01 129.077 0.00 673.13721.85 44.1771.14 0.1770.01

30 Lactobacillus casei 6.2970.01 5.7070.01 166.16717.00 104.3474.30 7.0673.25 0.0570.02

27 6.1170.01 5.3670.01 178.5374.37 190.8974.37 13.0675.82 0.0870.05

4 Lactococcus lactis ssp. cremoris 6.0070.07 4.3970.01 184.717 8.74 246.54717.48 5.2172.62 0.0470.02

6 6.2070.01 4.5570.01 159.9874.37 190.8978.47 0.00 0.0470.01

28 5.9470.01 4.4470.00 166.16717.48 104.3474.37 0.00 0.2470.02

51 5.9470.01 4.7270.00 122.8974.37 98.16726.23 11.1070.96 0.0570.02

52 6.0870.02 4.5270.01 159.9878.74 178.53739.34 9.2873.44 0.3070.01

Mean values7standard deviations are expressed as: pH after 8 and 24 h; mg/ml of Gly released after 24 h and 7 days; autolysis percentages (%) and

autolysis rates (AR).

L. Aquilanti et al. / LWT 40 (2007) 1146–11551152

be ascribed to the loss or gain of plasmids, since somecarbohydrate fermentation capacities are plasmid-encoded(Ahrne, Molin, & Stahl, 1989).

RAPD with a single primer followed by UPGMA clusteranalysis did not allow an efficacious discrimination of theisolates at the strain level. Although, a certain genotypicdiversity was found within each species, the finding of asame RAPD profile for isolates from different sourcesconfirmed the relatively low discriminating power of theRAPD typing technique (Tynkkynen, Satokari, Saarela,Mattila-Sandholm, & Saxelin, 1999), particularly when asingle primer is used. On the contrary, a high degree ofdiversity was observed in the technological traits assayed,as the tendency to lysis, in accordance with what has beenpreviously reported for Lactobacillus (Cibik & Chapot-Chartier, 2004) and Lactococcus (Ouzari, Cherif, & Mora,2002) strains. With respect to the above-cited trait, the

isolates belonging to the species Lb. brevis were the mostsuitable for use as starter cultures, since they showed thehighest percentages of lysis. Indeed, the tendency to lysis ofa starter culture is essential for the release of the bacterialintracellular enzymes into the cheese (Crow et al., 1995)and, in turn, for the increase of the cheese sensory andtextural properties (Sallami, Kheadr, Fliss, & Vuillemard,2004). Assessment of the amino-peptidase and dipeptidyl-peptidase activities, showed that these two technologicaltraits were highly dependent on the strain and thesubstrate, although in all cases the highest levels werefound on L-Lys- and L-Arg-L-Pro-p-NA. Notably (L-Lys)-aminopeptidase was seen in all of the strains, while a lackof activity towards L-Pro, L-Gly-L-Pro, L-Arg-L-Pro andL-Glu was seen for a few Lb. brevis and Lb. plantarum

isolates, although the X-Pro-dipeptidyl aminopeptidaseactivities are known to be active in all of the LAB species

ARTICLE IN PRESS

Table 4

Peptidase activities of the 33 lactic acid bacteria isolated from Canestrato Pugliese cheese towards L-lysine (L-Lys), L-glucine (L-Glu), L-proline (L-Pro), L-

glycine-proline (L-Gly–Pro), L-arginine-proline (L-Arg–Pro) p-NA

Isolate Species Peptidase activity

L-Lys-pNA L-Glu-pNA L-Pro-pNA L-Gly-L-Pro-pNA L-Arg-L-Pro-pNA

8 Lactobacillus brevis 4.5570.34 1.4170.24 0.00 9.1071.36 19.8971.02

14 17.2070.00 1.5170.19 1.0370.10 0.00 0.00

45 11.3070.30 0.4070.09 0.3470.05 3.4370.09 4.0570.91

46 47.2372.15 0.00 0.5370.02 12.0370.02 11.1270.22

48 16.8871.26 2.4870.38 3.3370.06 0.00 15.5571.89

68 14.9770.16 2.5370.08 0.00 3.9670.32 23.2371.62

77 12.0870.35 0.8170.03 0.00 8.1370.35 8.6370.35

81 73.8473.87 3.5571.16 0.00 3.2870.00 15.5871.55

83 16.1071.85 1.6970.06 8.7070.62 6.0971.23 26.9971.23

86 68.2671.21 1.3270.18 0.5170.07 0.00 19.6271.21

49 37.8670.75 0.0670.00 1.1770.30 17.5070.70 18.1370.00

58 23.4673.02 10.8770.30 13.5171.01 27.0272.01 18.4872.01

72 91.59715.54 2.6170.00 3.4470.10 43.5970.52 39.2071.55

73 12.6573.58 0.5070.10 0.0970.03 0.00 11.2071.53

78 45.5077.04 0.3070.03 1.0870.07 13.5170.00 2.1670.15

29 Lactobacillus plantarum 79.2372.87 0.1270.06 2.3370.43 12.2970.14 24.3775.75

33 12.6570.51 1.4170.15 0.6470.01 10.4870.51 18.0771.02

55 16.5470.35 0.1370.00 3.4470.10 43.5970.52 4.2370.92

23 14.7170.00 0.5570.08 0.00 0.00 27.5872.60

56 8.6370.36 0.7270.05 0.1070.02 5.2070.18 2.1070.25

59 8.7370.00 0.9770.14 0.4770.05 2.7270.05 3.6970.48

17 51.6471.06 0.8270.11 1.4270.11 15.6270.65 16.0970.53

18 24.3473.48 17.7770.77 4.5170.19 25.9871.93 42.3971.93

19 8.8771.21 2.2770.67 2.2470.71 21.3370.20 11.5670.61

35 57.0573.51 5.4570.70 8.1870.35 12.1570.35 24.8070.00

74 39.0570.00 0.3670.08 0.00 0.00 15.0270.00

30 Lactobacillus casei 57.0573.51 5.4570.70 8.1870.35 12.1570.35 24.8070.00

27 33.7772.51 7.1971.89 1.1570.38 12.3570.88 19.5572.51

4 Lactococcus lactis cremoris 29.7171.08 0.3870.05 0.5570.01 16.2570.07 19.0471.08

6 57.3571.98 2.0970.49 1.1570.35 13.2870.00 19.9371.48

28 14.5470.46 0.4370.09 0.6970.09 0.1970.00 0.00

51 10.6671.01 1.5670.20 0.3070.03 0.00 19.1971.01

52 27.7371.01 0.9470.13 0.7770.11 2.6370.30 5.7570.30

Mean values7standard deviation are expressed as absorbance at 410 nm.

L. Aquilanti et al. / LWT 40 (2007) 1146–1155 1153

(Kunji, Mierau, Hagting, Poolman, & Konings, 1996).Particularly, the strong X-Pro-dipeptidyl aminopeptidaseactivities of 58, 72 and 18 isolates make them of potentialinterest for use as starters, because of their capacity torelease large amounts of proline from caseins during thecheese ripening (Swaisgood, 1992).

From the results obtained in the present research work itemerged that although RAPD analysis has been widelyused for typing of LAB, the genotypic diversity highlightedthrough this technique is not necessarily related tobiochemical and metabolic differences among the isolates.This evidence underlines once again the usefulness of apolyphasic approach for the exhaustive characterizationof candidate strains to be used as starter cultures forcheese-making. Indeed, a combined approach that isbased on phenotypic and genotypic techniques increases

the resolution of each independent device, allowingthe detectable levels of strain heterogeneities to bewidened. The reliability of such an approach was alsodemonstrated by several studies focused on theevaluation of both the genetic diversity and technologicalproperties of LAB isolated from different cheeses(Lombardi et al., 2002; Giraffa et al., 2004) and dairyproducts (Mora et al., 2002).Based on the overall results from such a polyphasic

approach, it emerged that none of our isolates showed thebest performances in all of the assays carried out. As aconsequence, it can be concluded that an appropriatemixture of a few isolates, each showing one trait of interest,could be used, also in view of the high heterogeneity of thewild lactic acid populations detected in this traditionalcheese.

ARTICLE IN PRESS

Table 5

The RAPD clusters and biotypes, related to the 33 lactic acid bacteria

isolated from Canestrato Pugliese cheese

Isolate Farm RAPD Phenotypic characterization

AA PA AR Biotype

8 F1 Lbb 1 a c a Lbr A

83 F1 Lbb 2 b c a Lbr B

81 F1 e c a Lbr C

72 F3 Lbb 3 e c a

58 F2 b b a Lbr D

73 F3 f c a Lbr E

78 F3 g c a Lbr F

77 F1 c a a Lbr G

68 F1 d c a Lbr H

45 F1 Lbb 5 d c a

46 F1 b c a Lbr I

48 F1 b c a

49 F2 c c a Lbr M

14 F1 e c a Lbr N

86 F1 Lbb 4 e c a

55 F1 Lbp 1 a a c LbpA

17 F3 Lbp 2 b b c Lbp B

74 F3 c a bc Lbp C

18 F3 Lbp 3 c c c Lbp D

19 F3 d a c Lbp E

33 F1 e d c Lbp F

35 F3 d c c Lbp G

56 F2 g a c Lbp I

59 F2 h a a Lbp L

23 F2 Lbp 4 i a c Lbp M

29 F1 Lbp 5 l c c Lbp N

30 F1 Lbc 1 a a a Lbc A

27 F3 Lbc 2 b a a Lbc B

4 F2 Lcc 1 bc b c Lcl A

6 F3 Lcc 2 a b c Lcl B

28 F3 c b b Lcl C

51 F3 c a c Lcl D

52 F3 b b a Lcl E

The statistical evaluation of the biotypes data related to: acidifying

activities after 8 h (AA); proteolytic activities after 24 h (PA) and autolysis

rates (AR). Within each species and data set, the isolates labelled with the

same letters are not significantly different (Po0.05).

L. Aquilanti et al. / LWT 40 (2007) 1146–11551154

Acknowledgements

The present research work was commissioned by themunicipality of Manfredonia (Foggia, Italy), within ProjectNo. 12/cluster C08-B ‘Valorization of dairy products’,supported by MURST. Thanks are due to ProfessorMarina Pasquini (Department of Food Science, Polytech-nic University of Marche, Italy) for the valuable statisticalelaboration of the phenotypic data.

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