International Journal of Food Microbiology 125 (2008) 330–335

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

Raw milk traditional Italian ewe cheeses as a source of Lactobacillus casei strains withacid-bile resistance and antigenotoxic properties

Aldo Corsetti a, Giovanna Caldini b, Mario Mastrangelo a, Francesca Trotta b,Sara Valmorri a, Giovanni Cenci b,⁎a Dipartimento di Scienze degli Alimenti, Università di Teramo, Via C.R. Lerici 1, 64023 Mosciano S. Angelo (TE), Italyb Dipartimento Biologia Cellulare e Ambientale, Università di Perugia, Via del Giochetto, I-06126, Perugia, Italy

⁎ Corresponding author. Dipartimento Biologia CelluMicrobiologia, Università di Perugia, Via del Giochetto,075 585 7333; fax: +39 075 585 7328.

E-mail address: gcenci@unipg.it (G. Cenci).

0168-1605/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2008.04.009

A B S T R A C T

A R T I C L E I N F O

Article history:

Twenty-five Lactobacillus ca Received 16 January 2008Received in revised form 18 April 2008Accepted 23 April 2008

Keywords:Acid-bile toleranceAntigenotoxicityLABLactobacillus casei

sei group strains isolated from ewe cheeses from Abruzzo region, central Italy,were identified by 16S rRNA gene sequencing, differentiated by RAPD-PCR analysis and characterized as invitro for acid-bile tolerance and antigenotoxic properties. All the strains were very susceptible to simulatedgastric fluid (pH 2.0) but most of them recovered viability (ca. 2–3 log-units) when transferred andmaintained in simulated intestinal fluid (0.5% w/v bovine bile) for 3 h. Some strains showed potential fordeactivating representative genotoxins as highlighted by the SOS-Chromotest. Twelve were active and ninemoderately active against 4-nitroquinoline-1-oxide, and one active and only one moderately active againstN-methyl-N′-nitro-N-nitrosoguanidine. The active strains produced evident spectroscopic modification ofgenotoxins after co-incubation. Most isolates with antigenotoxic activity resulted as acid-bile tolerantdemonstrating that cheese autochthonous lactobacilli may reach the gut as a viable form and preventgenotoxin DNA damage.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Lactobacilli occur naturally in various fermented products and inmany cases represent the dominating microflora (De Angelis et al.,2001; Corsetti and Settanni, 2007). In ripened cheeses, adventitiousmesophilic lactobacilli, referred to as non starter lactic acid bacteria(NSLAB), are usually present because of post-pasteurization contam-ination but may also constitute part of the raw milk microflora and,surviving pasteurization, reach 107–108 cfu g− 1 (Mannu et al., 2000;De Angelis et al., 2001). The indigenous microbial contents of cheesescould be considered some of the main factors in determining thetypical cheese features and heterogeneous NSLAB constitute a greatpart of the autochthonous microbial community commonly includingsome species of the Lactobacillus casei group (e.g. L. casei, Lactobacillusparacasei subsp. paracasei, L. paracasei subsp. tolerans) (De Angeliset al., 2001; Coueret et al., 2004; Švec et al., 2005).

Different studies have been aimed to the investigation offunctional properties of fermented foods as well as of autochthonouslactobacilli in in vitro research and, recently, also focused onantimutagenic and antigenotoxic properties (Vizoso Pinto et al.,

lare e Ambientale, LaboratorioI-06126 Perugia, Italy. Tel.: +39

l rights reserved.

2006). As reported by Maron and Ames (1983) a close correlationexists between mutagenesis and carcinogenesis.

A variety of genotoxic food related compoundsmay be found in theintestinal tract including “primary naturally occurring foodmutagens”(e.g. mycotoxins and plant glycosides), “secondary food mutagens”(e.g. polycyclic aromatic hydrocarbons and aromatic heterocycleamines) and “endogenous compounds” (e.g. secondary bile acidsand nitrosamines) (Cenci et al., 2005). Thus, the inhibition of DNAgenotoxins is thought to be an important tool for reducing gutpathologies and colon cancer incidence.

Lin and Chou (2006), by studying the extracts of the soybean koji,demonstrated that fermentation resulted as an enhanced antimuta-genic effect on the activity of the reference genotoxin 4-NQO.Moreover, the lower rate of breast, prostate, and colon cancer inAsian countries has been attributed to the consumption of soy basedfoods containing antimutagenic factors (Barnes et al., 1990). Otherauthors also stated that a diet rich in antimutagens can reduce theincidence of cancer in humans and indicated that the use ofantimutagens and anticarcinogens in daily life is the most effectiveway to prevent human cancer and genetic disease (Kim et al., 2000). Atpresent, relevant data have been accumulated supporting thehypothesis of bacterial activity in decreasing the risk of colon cancer(Massi et al., 2004). In particular, lactic acid bacteria and bifidobacteriashow antimutagenic activities against heterocyclic amines, N-nitrosocompounds, benzo [a] pyrene and aflatoxin B (Hsieh and Chou, 2006)

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and fermented milk containing lactic acid bacteria has also shown topossess antimutagenic or anticarginogenic activity (Nadathur et al.,1995). Moreover both epidemiological and in vitro studies supportevidence of antigenotoxicity in some probiotic strains implying thereduction of genotoxic compounds which act as tumour initiators(Cenci et al., 2005). Very few studies have been done on theantigenotoxic–antimutagenic properties of autochthonous lactobacillirepresenting the prevailingmicroflora inmany ripened Italian cheeses(Caldini et al., 2008).

In this paper 25 strains belonging to the L. casei group isolatedfrom Italian cheeses produced with raw milk (Pecorino cheese) werestudied for the inhibition of two potent DNA-reactive agents. More-over, since those microorganisms should act against genotoxiccompounds in the intestine after surviving the gastro-intestinalhurdle, the resistance to gastric acidity and to bile salts was evaluatedas well.

The aim of the paper is to show that certain autochthonouslactobacilli of cheeses have got functional properties already demon-strated in probiotic bacteria, such as acid-bile tolerance andantigenotoxic activity. Since NSLAB are present in high numbers insome traditional Italian cheeses, it could be of interest to verify iftraditional fermented foods, normally consumed as part of a daily dietcan be assimilated for some properties of their microflora, tofunctional foods like fermented milk containing probiotics.

2. Materials and methods

2.1. Bacterial strains, media and identification

The 25 lactobacilli considered in this study were isolatedpreviously from central Italy (Abruzzo region)-artisan Pecorinocheeses (“Pecorino d'Abruzzo” cheese), following the procedurereported by De Angelis et al. (2001). All the cheeses were producedby using raw ewe milk without starter cultures and were analysedwhen they were ready for market (ripening time ranging from 45 to60 days). All the isolates were stored as pure cultures, labelled fromMSA 1 to MSA 25, at − 80 °C in the Culture Collection of theDepartment of Food Science—University of Teramo. At the occurrencethe strains were propagated in MRS (Oxoid, Basingstoke, UK) broth at30 °C for 24 h. The strains were phenotypically characterized bymorphological, physiological and biochemical assays, including sugarfermentation by using the API 50 CHL system (bioMerieux, Marcy-l'Etoile, France) as reported by De Angelis et al. (2001).

For genotypic identification microbial DNAs were purified accord-ing to the method reported by De Los Reyes-Gàlivan et al. (1992) from2 mL samples of overnight cultures and used for the 16S rRNA genesequence analysis using LacbF/LacbR primer (MWG Biotech AG,Ebersberg, Germany) pair according to the protocol reported byCorsetti et al. (2004). PCR products were purified by the GFXTM PCRDNA and the Gel Band Purification Kit (Amersham Biosciences,Piscataway, NJ, USA). DNA sequencing reactions were performed byMWG Biotech AG. The isolates, besides L. casei DSM 20011T (ATCC393T) were also characterized by RAPD (Randomly AmplifiedPolymorphic DNA)-PCR using the primer M13. Escherichia coli PQ37(sfiA::lacZ, uvrA, rfa, Phoc), used as tester strain in the SOS-Chromotest, was supplied by the Institute Pasteur (Paris, France).PQ37 was grown in L-broth (10 g L−1 tryptone, 5 g L−1 yeast extract,10 g L−1 NaCl) and La-broth (supplemented with 20 μg mL−1

ampicillin). All products for media preparation were obtained fromOxoid.

2.2. Chemicals

O-nitrophenyl-β-D-galactopyranoside (ONPG) and p-nitrophenyl-phosphate (PNPP), for evaluation of β-galactosidase and alkalinephosphatase activity respectively, were purchased from Sigma-

Aldrich (St Louis, MO, USA). Bovine bile (B-8381, Sigma-Aldrich),containing free and conjugated bile acids [1:1 w/v] were dissolved indistilled water, filtered through a 0.45 μm membrane (Sartorius,Göttingen, Germany) and added to a sterile saline. Pepsin (P-7000,porcine stomach mucosa) and pancreatin (P-8096, porcine pancreas)were purchased by Sigma-Aldrich.

2.3. Genotoxins

Two direct-acting compounds were used: 4-nitroquinoline-1-oxide (4-NQO, CAS no. 56-57-5) and N-methyl-N′-nitro-N-nitroso-guanidine (MNNG, CAS no. 70-25-7), both purchased from Sigma-Aldrich. Stock solutions (1 mg mL−1) were prepared, for 4-NQO indimethyl sulphoxide and for MNNG in saline, and stored at − 20 °C.Working solutions were prepared in saline just before testing.

2.4. Simulated gastro-intestinal condition

Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF)were prepared fresh daily in order to evaluate the acid-bile toleranceof the 25 strains. Lactobacilli were suspended in SGF (pH 2.0)containing pepsin (1 mg mL−1), and maintained at 37 °C for 2 h.After acid preincubation cells were centrifuged (6000 ×g, 15 min),resuspended in SIF (pH 7.4) containing pancreatin (1 mg mL−1) andbovine bile (0.5% w/v) dissolved in saline, and maintained at 37 °C for3 h (Haller et al., 2001; Cenci et al., 2006). After acid and bilechallenges samples were removed, serially diluted, and plated onMRSagar to determine cell viability.

2.5. Genotoxicity inhibition assay

Stationary phase cells of lactobacilli (grown in MRS broth for 24 hat 30 °C) were washed and resuspended in saline (109 cfu mL−1). Insome cases cell concentration was examined for antigenotoxicity inthe range of 105–109 cfu mL−1. Genotoxin (4-NQO or MNNG) was thenadded (0.1 mM) and co-incubation was maintained under agitation(150 min at 30 °C). The recovered supernatant (6000 ×g, 15 min) wasthen filtered by 0.45 μm membrane (Sartorius) and residualgenotoxicity was detected using SOS-Chromotest, which is a primaryDNA-damage assay on E. coli (Quillardet and Hofman, 1993). With thepresence of DNA damage the tester strain (E. coli PQ37) activates theSOS-response which, due to sfiA::lacZ fusion, induces β-galactosidase(BG) activity.

The constitutive alkaline phosphatase (AP) production of PQ37 isan indicator of protein synthesis in the presence of sub-lethal doses ofgenotoxin. The SOS induction factor (IFsos) is defined as BG to the APratio of the sample under analysis, divided by the same ratio ofnegative control (without genotoxin). The enzyme activities weredetected colorimetrically at 405 nm using ONPG and PNPP assubstrates, respectively. Enzyme activities were expressed as conven-tional units (U = A405×1000/t, where t indicates the substrateconversion time in minutes). PQ37 was grown overnight in La-brothand then transferred (100 μL) into 5 mL of the same medium andincubated for 2 h (OD520: 0.3–0.4). One mL of culture was then addedto 9 mL of fresh LB-broth and fractions of 600 μL were mixed with20 μL of sample containing genotoxin (or supernatant of genotoxin-treated culture) and then incubated for 150 min at 37 °C. The sameprocedure was used to determine the antigenotoxicity of heat killed(110 °C, 30 min) cell.

The genotoxicity inhibition (GI) produced by different strains wasalso given in percentages considering the IFsos of positive controls(genotoxin without lactobacilli co-incubation) as 100%. The thresholdfor strain antigenotoxicity was fixed as follows: GIN75% active, GIbetween 25 and 75% moderately active, GIb25% inactive. Theexperiments were carried out in the absence of exogenous metabolicactivation of genotoxins.

Fig. 1. UPGMA dendrogram showing the relationships among 25 strains of the Lactobacillus casei group plus L. casei ATCC 393T, obtained by RAPD patterns with M13.

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2.6. Physico-chemical analyses

Supernatants of genotoxin-treated cultures were examined byabsorbance profiles (Shimadzu UV-1204 spectrophotometer) in orderto prove chemico-biological interactions.

3. Results

3.1. Identification of the isolates and strain typing by RAPD-PCR

All the 25 strains selected for this studywere gram-positive, catalasenegative and rod shaped. Phenotypic characterizationbasedona growthrate at 15 and 45 °C, CO2 production from glucose, NH3 production from

Fig. 2. The survival of Lactobacillus casei strains to consecutive exposure to simulated gastric flMRS agar from two experiments. Bars indicate the standard deviation (SD).

arginine and esculin hydrolysis (Hammes and Vogel,1995), biochemicalprofiles obtainedby theAPI 50CHkit, andgenotypic identificationbasedon 16S rRNA gene sequencing, classified the 25 lactobacilli as belongingto the L. casei group. RAPD-PCR fingerprints generatedwithM13 primershowed complex bandpatterns, ranging fromaminimumof 4 bands forstrains MSA12 and MSA 25 to a maximum of 11 bands for strain MSA9and produced the dendrogram shown in Fig. 1 indicating a highbiodiversity among the strains.

3.2. Tolerance to gastro-intestinal transit

The effect of simulated gastro-intestinal transit is presented in Fig.2. All the strains were highly susceptible to low pH showing a marked

uid (SGF) and simulated intestinal fluid (SIF). Data are shown as means viable counts on

Fig. 3. The tolerance of Lactobacillus casei strains to consecutive acid (SGF) and bile (SIF)challenge. Data represent viable cell counts (log10 cfu mL−1) of each strain after thechallenges. The vertical and horizontal dashed lines show the means value (arbitrarythreshold to distinguish the relative strain tolerance) of cell count in SGF and SIF,respectively. MSA strains: (A) 3, 5, 9,11,19,15, 25, 29 (B) 1, 4, 8, 10,12,13,14,17, 20, 22, 25(C) 7, 18, 21, 24 (D) 7, 16.

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decrease of cell viability after the exposure to simulated gastric fluid.With reference to control cultures, without stress, the treatedsuspensions showed an average reduction (±SD) of 4.42±1.32 log-units (range 1.41–6.28). Even in the presence of a relevant reduction inculture viability, the viable counts of different strains estimated after

Fig. 4. In vitro antigenotoxicity of Lactobacillus casei strains towards 4-NQO (A) and MNNG (B150 min. Percent inhibition was calculated from residual activity (SOS induction factor) evaluthreshold levels for antigenotoxicity (see Materials and methods). (●) Strainwith a marked sBars indicate the standard deviation (SD).

the challenge at pH 2.0 were comparable and all the examinedlactobacilli survived the injury miming the gastric environment.

An evident recovery of viability (on average 1.94±1.40 log-units;range 0.98–5.08) was observed when the cells were transferred andmaintained in simulated intestinal fluid for 3 h after the acidicpreincubation. Only one strain (MSA16) showed no increase of viablecount in passing from SGF to SIF. The recovery of viability in simulatedintestinal fluid was also appreciable for 8 strains which were verysensitive to acid stress (Fig. 3, plot A). Within the 25 strainsconsidered, at the same time 11 were tolerant to acid and bile (Fig.3, plot B).

3.3. Antigenotoxic properties

The inhibitory activity of the strains against the referencegenotoxins is shown in Fig. 4. As far as 4-NQO, the strain's behaviourwas as follows: 12 produced high inhibition, 9 were moderately activeand 4 inactive. The same results were obtained when testing the cellconcentration in the range of 105–109 cfu mL−1. Relative toMNNG only1 strain was highly active, 13 moderately active and 11 inactive. Theantigenotoxic effect was eliminated when the lactobacilli were heattreated (110 °C, 15 min) before their co-incubation with genotoxinsand no antigenotoxic activity was observedwhen the genotoxins weremixedwith cell-free supernatants from active strain cultures (data notshown).

3.4. Genotoxin modification by bacteria

The antigenotoxicity originated by L. casei was strain dependentand some relevant spectroscopic modifications of the genotoxins were

) in the SOS-Chromotest. Strains were co-incubated with 0.1 mM genotoxin at 30 °C forated on supernatants in relation to that of a positive control. The dashed lines show theurvival ability after acid-bile challenge. Data are shown as means from two experiments.

Fig. 5.UV spectra of genotoxins after co-incubationwith L. casei strains which produced and did not produce genotoxicity inhibition (GI). A, 4-NQO plus MSA20 (GI=90.4%); B, 4-NQOplus MSA16 (GI=0%); C, MNNG plus MSA20 (GI=77.3%); D, MNNG plus MSA16 (GI=0%). The dashed profile represents genotoxin spectrum without bacteria co-incubation.

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observed after their co-incubation with active strains. The maximumabsorbance peak of both 4-NQO and MNNG was shifted to a shortwavelength (hypsochromic shift), compared to the normality typicalof the compounds. The hypsochromic shifts were from 10 to 20 nmand from 6 to 13 nm for 4-NQO and MNNG, respectively. Therepresentative UV spectra of genotoxins after co-incubations with L.casei strains which produced (e.g. strain MSA20) and did not produce(e.g. strain MSA16) inhibition of their activity are shown in Fig. 5.

4. Discussion

The dominant non starter lactic acid bacteria (NSLAB) found indifferent type of ripened cheeses commonly include L. casei/paracaseistrains (Coueret et al., 2004; De Angelis et al., 2001), a group oflactobacilli for which a clear-cut taxa is difficult to reach (Vasquez etal., 2005). The poliphasic (phenotypic and genotypic) approachapplied in the present study confirmed such a finding and classifiedthe 25 strains previously isolated from artisan cheeses produced withraw ewe milk (“Pecorino d'Abruzzo” cheese) as L. casei/L. paracaseisubsp. paracasei, without clearly discriminating between the twospecies. Recently, Švec et al. (2005) reported a close similarity amongL. casei and L. paracasei subsp. paracasei dairy strains after a taxonomicstudy based on ribotyping with EcoRI and HindIII. Moreover, Massiet al. (2004), studying lactobacilli isolated from human gut and vagina,found a high similarity between L. casei LC10 and L. paracasei subsp.paracasei ribotype reference pattern from the RiboPrinter database.Our results are also in accordance with other studies supporting thesynonymy between of L. casei and L. paracasei (Švec et al., 2005).

Therefore, we refer to 25 lactobacilli in this paper as L. casei groupstrains. Since strains selected for particular functional properties,which fit the principal aim of this study, have to be clearlycharacterized below the species level (Parker, 1974), a DNA-basedtyping method was applied to differentiate the 25 L. casei groupstrains at the clonal level.

Strain typing with M13 RAPD-PCR pointed out high biodiversity inthe L. casei group. In particular, by considering an arbitral similaritylevel around 50%, the 25 strains clustered in 5 different groups. Thus,besides a high genomic variability into each cluster, lactobacillioccurring in different clusters may be regarded as more dissimilarstrains.

Acid and bile tolerance represent basic in vitro selection criteria forprobiotics (Vijendra and Prasad, 2005; Morelli, 2007) and, in our case,a fundamental prerequisite for putative in situ functionality of strainswith antigenotoxic properties.

Miming gastro-intestinal transit, we clearly highlighted that thebacterial stress originated by low pH may be overcome after thesubsequent treatment in presence of bile. The rapid viability increaseafter 3 h in SIF can depend on the fact that a great amount of cells wasable to recover from viable-but-non-culturable condition caused bythe SGF treatment. After the acid challenge the survival fractionobserved for L. casei strains at pH 2 resulted higher than thatdescribed for the probiotics L. acidophilus LA-1 and L. rhamnosus GG(Prasad et al., 1998), for some probiotic L. casei and for starter lacticacid bacteria (L. delbrueckii subsp. bulgaricus, Lactococcus lactis,Streptococcus thermophilus) (Vinderola and Reinheimer, 2003). Thevalues found for the strains characterized in this study correspond tothat observed for L. paracasei isolated from fermented foods (Haller etal., 2001) and for some probiotic strains of L. acidophilus and B. bifidum(Vinderola and Reinheimer, 2003).

The cell counts observed after treatment with simulated intestinalfluid were lower when compared with those reported for lactobacilliand bifidobacteria of different origin (Charteris et al., 1998), but all thestrains tested exhibited very good tolerance to 0.5% w/v bile, a dosehigher than that usually recommended (0.3% w/v) in evaluatingprobiotic characteristics. Moreover, with only one exception, all thestrains showed a moderate growth at the above bile concentrationafter a prolonged incubation in SIF (data not shown), probably due totheir capability to metabolize conjugated bile salts.

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In the framework of probiotic multifunctionality, some papersfocused on starter and probiotic lactic acid bacteria antigenotoxicity(Cenci et al., 2002; Orrhage et al., 2002).

Recently, our group pointed out for the first time that this propertyis also shown by some cheese non starter lactobacilli, including L.rhamnosus, L. casei, Lactobacillus plantarum, L. brevis and Lactobacillusspp. isolated from artisan, raw milk ewe cheeses, produced in centralItaly (Umbria region) (Caldini et al., 2008). In the present study, sinceewe cheeses are traditionally consumed throughout Italy and due tothe increasing interest of L. casei, both as probiotic and starter adjunct(Phillips et al., 2006), we specifically addressed our interest to thecombined evaluation of both gut resistance and antigenotoxicproperties in autochthonous strains within the L. casei group thatwas isolated from rawmilk ewe cheeses produced in another region ofcentral Italy (Abruzzo region).

We showed that antigenotoxic activity is strain dependent, relatedto the dualism strain-genotoxic compound and is not influenced byviable cell concentration in the range 105–109 cfu g−1 considered inthis study. The first finding is in accordance with the current evidencethat suggests that specific probiotic properties are strain dependent(Holzapfel et al., 2001). The cell-density dependent antigenotoxicitywas, on the contrary, pointed out by other authors investigating thereduction of faecal water genotoxicity by the probiotic strains of Bifi-dobacterium spp. and L. plantarum (Burns and Rowland, 2004). On theother hand, antigenotoxicity was strongly or completely reducedwhen the compounds were co-incubated with dead cells, instead oflive cells (data not shown).

With relation to the different behaviour of a same strain againstthe two reference genotoxins evaluated in this study, even if adifferent mechanism of the genotoxin can explain such a finding, thedetoxification mechanism implies, in all cases, a chemical modifica-tion of the toxic compound. Studies are in progress in order to identifythe inactive compounds.

5. Conclusions

This study provides first hand evidence about the implication ofcheese autochthonous L. casei group strains on the deactivation ofmodel genotoxins. Interestingly, strains with antigenotoxic propertiesalso showed acid-bile tolerance, indicating that indigenous micro-organisms dominating some traditional, ripened Italian cheeses,might reach the gut as viable cells and prevent DNA damage asdemonstrated for both probiotics and starter bacteria of dairyindustry.

Further studies are warranted in order to evaluate the viability andthe technological characteristics of the most interesting strainsselected in this study in view of development in cheeses with newfunctional properties.

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