Antagonistic activity against foodborne-pathogens of lactic acid bacteria from Calabrian raw milk cheeses

ANTAGONISTIC ACTIVITY AGAINST FOODBORNE PATHOGENS OF LACTIC ACID BACTERIA FROM

CALABRIAN RAW MILK CHEESES

The artisanal raw milk cheeses made without the addition of lactic acid bacteria (LAB) starters are very consumer attractive since intensely flavoured, due to the action of endogenous microflora; however, they could imply hygienic and health concerns for the possible transmission of foodborne pathogens. Really, ruminants are an important reservoir of verocitotoxic Escherichia coli (VTEC),

Salmonella spp., Listeria monocytogenes, Staphylococcus aureus and others, resulting in a possible contamination of their products (La Torre et al., 2007; Normanno et al., 2007a; 2007b; Normanno,

2007). Strategies based both on use of starters and microbial cooperation and antagonism allow obtaining improved safe foods. LAB bacteriocins contribute to the inhibition of foodborne pathogens such as Listeria monocytogenes (Rodriguez et al., 2001), Bacillus cereus (Morgan et al., 2001), Staphylococcus aureus (Olasupo et al., 1999), and VTEC Escherichia coli O157:H7 (Rodriguez et al., 2005). Caprino d’Aspromonte, Pecorino d’Aspromonte, and Pecorino del Poro cheeses, made from raw goats’ and ewes’ milk respectively, without the addition of LAB starters, are widespread in the Calabrian area (Southern Italy). Their ripening depends on spontaneous microflora and the raw milk could represent a favourable environment for foodborne pathogens to survive and grow. Previous studies on these cheeses have led to identifying of strains of Lactobacillus paracasei subsp. paracasei able to inhibit dairy Escherichia coli (Caridi, 2002; 2003).

AIM OF THE WORK

Aim of this work was to select foodborne pathogen-inhibiting strains among the endogenous LAB isolated from the artisanal Calabrian raw milk cheeses.

INTRODUCTION

REFERENCES

1. La Torre L., Parisi A., Fraccalvieri R., Normanno G., Nardella La Porta M.C., Goffredo E., Palazzo L., Ciccarese G., Addante N., Santagada G. (2007) Low prevalence of Listeria monocytogenes in foods from Italy. Journal of Food Protection 70, 1507–1512.

2. Normanno G., La Salandra G., Dambrosio A, Quaglia N.C., Corrente M., Parisi,A., Santagada G., Firinu A., Crisetti E., Celano G.V. (2007a) Occurrence, characterization and antimicrobial resistance of enterotoxigenic Staphylococcus aureus isolated from meat and dairy products. International Journal of Food Microbiology 115, 290–294.

3. Normanno G., Corrente M., La Salandra G., Dambrosio A., Quaglia N.C., Parisi A., Greco G., Totaro M., Virgilio S., Celano G.V. (2007b) Methicillin-resistant Staphylococcus aureus (MRSA) in foods of animal origin product in Italy. International Journal of Food Microbiology 117, 219–222.

4. Normanno G. (2007) Current status on the etiology, epidemiology, food safety implications and control measures in Escherichia coli O157:H7 infections. In: Food Microbiology Research Trends, Marta V. Palino Ed., Novascience Publisher, N.Y., 4, 131–154.

5. Rodriguez E., Arquès, J.L., Gaya, P., Nunez, M., Medina, M. (2001) Control of Listeria monocytogenes by bacteriocins and monitoring of bacteriocin-producing lactic acid bacteria by colony hybridization in semi-hard raw milk cheese. Journal of Dairy Research 68, 131– 137.

6. Morgan S.M., Galvin, M., Ross, R.P., Hill, C. (2001) Evaluation of a spray-dried lacticin 3147 powder for the control of Listeria monocytogenes and Bacillus cereus in a range of food systems. Letters in Applied Microbiology 33, 387–391.

7. Olasupo N.A., Schillinger, U., Narbad, A., Dodd, H., Holzapfel, W.H. (1999) Occurrence of nisin Z production in Lactococcus lactis BFE 1500 isolated from wara, a traditional Nigerian cheese product. International Journal of Food Microbiology 53, 141–152.

8. Rodriguez E., Calzada, J., Arquè, J.L., Rodriguez. J.M., Nunez, M., Medina, M. (2005) Antimicrobial activtity of pedici-producing Lactococcus lactis on Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 in cheese. International Dairy Journal 15, 51–57.

9. Caridi A. (2002 ) Selection of Escherichia coli-inhibiting strains of Lactobacillus paracasei subsp. paracasei. Journal of Industrial Microbiology & Biotechnology 29, 303–308.

10. Caridi A. (2003) Identification and first characterization of lactic acid bacteria isolated from the artisanal ovine cheese Pecorino del Poro. International Journal of Dairy Technology 56, 105–110.

11. Spelhaug S.R., Harlander, S.K., (1989) Inhibition of food-borne bacterial pathogens by bacteriocins from Lactococcus lactis and Pediococcus pentosaceous. International Journal of Food Protection 52, 856–862. 12. European Commission, 2005. Commission Regulation (EC) No _{2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Union L 338, 1–26.}

MATERIALS AND METHODS

Seven Pecorino del Poro and one Pecorino d’Aspromonte ewes’ cheeses were sampled from dairy farms located in the province of Vibo Valentia; four Caprino d’Aspromonte goats’ cheeses were sampled from dairy farms located in the province of Reggio Calabria. Cheeses were made using raw milk without the addition of starters according to traditional procedures.

Cheese samples (10 g) were homogenized in 90 ml of sterile physiological saline for 2 min in a blade homogenizer and filtered through sterile gauze. Aliquots of 1 ml were 10-fold diluted in physiological sterile saline to enumerate LAB and E. coli and to isolate different microbial groups. Lactobacilli were isolated on Man-Rogosa-Sharpe (MRS) agar and incubated anaerobically by Gas Pack catalysts AnaeroGen 2.5L at 30 °C for 3 days. Coccal-shaped LAB were isolated on M17 agar and incubated anaerobically at 30 °C for 3 days. Dairy E. coli strains were isolated on Petrifilm 3M and incubated aerobically at 37 °C for 24 h. Working cultures were grown on broth media MRS, M17, and Lactose Broth for lactobacilli, lactococci, and E. coli strains, respectively. One strain of the following foodborne pathogens were used for the evaluation of the antagonistic activity of LAB against them: VTEC E. coli O157:H7 (VT1 + VT2 producer), VTEC E. coli O26 (VT1 producer), L. monocytogenes, Salmonella spp. 1023, and Salmonella Typhimurium, and enterotoxigenic Staphylococcus aureus (SEA- SEC producers). Further, E. coli ATCC 45922, not pathogenic strain, was used as control strain. With the exception of the ATCC strain, all the strains used were isolated from milk or dairy products, using standard methods.

The LAB isolated were screened for the antagonistic activity against five dairy E. coli strains and control strain E. coli ATCC 45922; later, against the different foodborne pathogens above mentioned, using the spot method (Spelhaug et al., 1989). Other than the LAB isolated, two Lactobacillus paracasei subsp.

paracasei, previously selected (Caridi, 2002), were tested.

A 24 h culture of each LAB was 10-fold diluted in 10 mmol/l Tris-HCl (pH 7.0), and 1 μl aliquots were spotted in triplicate onto MRS agar. Plates were incubated anaerobically for 24 h and then overlaid either with Lactose soft agar (0.7% agar) or Triptic Soy soft agar (0.7% agar) seeded, respectively, with 0.1 ml of overnight cultures of E. coli strains and different foodborne pathogens. After additional 18 h, plates were checked for inhibition zones around spots of the presumed producers. The inhibition zones were measured as mm by Autodesk Autocad 2007. All the trials were carried out in duplicate.

RESULTS

LAB ranged from 4 x 107_{cfu/g to 3 x 10}10_{cfu/g in the cheeses tested; moreover, E. coli}

were not revealed or present until a maximum of 1.2-1.4 x 106_{cfu/g. Excluding the last}

case, with only two values over the limit foreseen by the 2005/2073 EC Regulation (EC, 2005). These results could be positively considered from the safety and healthy point of view. The finding of cheeses with low values of E. coli could be due to: 1. good hygienic quality of milk; 2. good hygienic conditions during cheese manufacturing; 3. presence of bacteriocine-like compound produced by LAB - our case.

Among the sixty-eight LAB isolated, 17 and 51 were coccal and rod-shaped, respectively. No coccal-shaped LAB possessed antagonistic activity against the five dairy E. coli strains isolated and the control strain E. coli, while LAB rod-shaped did. The best seven strains of Lactobacillus spp., together with two L. paracasei subsp.

paracasei – listed in Table 1 - were tested for their antagonistic activity against the

foodborne pathogens. Figure 1 shows the inhibition zone of LAB strains against pathogens.

Table 2 highlights the antagonistic activity of the strains based on their inhibition zones expressed as mm. All the Lactobacillus strains showed antagonistic activity against S. Typhimurium - with a minimum of 5.22 mm for strain Lb23PP and a maximum of 12.47 mm for strain Lb07PP - and Salmonella spp. 1023 - with a minimum of 5.20 mm for strain Lb02E and a maximum of 10.77 mm for strain Lb07PP. The strains Lb06PP, Lb07PP, Lb20PP, Lb21PP, and Lb23PP showed antagonistic activity against VETC E. coli O157:H7 with a minimum of 4.46 mm for strain Lb21PP and a maximum of 7.06 mm for Lb23PP. The strains Lb06PP, Lb07PP, Lb17PP, Lb21PP, and Lb23PP showed antagonistic activity against VETC E. coli O26 with a minimum of 4.73 mm for strain Lb06PP and maximum of 8.08 mm for strain Lb23PP. All the strains tested showed antagonistic activity against enterotoxigenic S. aureus and L. monocytogenes; for the latter pathogen it was not possible to determine the inhibition zones as mm since it grows as thin layer visible only by naked eyes. Therefore, for L. monocytogenes the results are based only on the visual assessment. Eight out nine strains showed antagonistic activity against E. coli ATCC 45922 with a minimum of 4.03 mm for strain Lb02E and a maximum of 9.87 mm for strain Lb05CA.

PERSPECTIVES

This study highlights the intense and different antagonistic activity induced by autochthonous lactobacilli against various foodborne pathogens, due presumably to production of bacteriocin-like compounds.

Moreover, the specie L. paracasei subsp. paracasei, for which the antagonistic activity against dairy E. coli, was already demonstrated was found active also against the control strain E. coli ATCC 45922 and against S. Typhimurium, Salmonella spp. 1023, S. aureus, and L.

monocytogenes.

Using selected LAB strains as adjunct cultures could be an interesting and effective strategy to protect artisanal raw milk cheeses by foodborne pathogens.

A. CARIDI

1

, M. GERIA

1

, R. SIDARI

1

, A. DAMBROSIO

2

, V. LORUSSO

2

, G. NORMANNO

2

1

Department of Scienze e Tecnologie Agro-Forestali e Ambientali (DISTAFA), "Mediterranea" University of Reggio

Calabria, Via Feo di Vito, I-89122 Reggio Calabria, Italy

2

Department of Health and Animal Welfare, University of Bari, S.P. Casamassima Km. 3, 70010, Valenzano (BA), Italy

Strains Specie Origin

Lb01PP Lactobacillus paracasei subsp.

paracasei Pecorino del Poro

Lb02E Lactobacillus paracasei subsp.

paracasei Pecorino del Poro

Lb05CA Lactobacillus spp. Caprino d'Aspromonte

Lb06PP Lactobacillus spp. Pecorino d’Aspromonte

Lb07PP Lactobacillus spp. Pecorino d’Aspromonte

Lb17PP Lactobacillus spp. Pecorino del Poro

Lb20PP Lactobacillus spp. Pecorino del Poro

Lb21PP Lactobacillus spp. Pecorino del Poro

Lb23PP Lactobacillus spp. Pecorino del Poro

Table 1 – Lactobacillus strains tested for the antagonistic activity against

foodborne pathogens

*Standard Deviation **Visual Assessment

Table 2 – Antagonistic activity of the strains based on their inhibition zones expressed as mm

Figure 1 – LAB antagonistic activity evaluated by spot method. Activity of strain Lb06PP against S. aureus (a )

and against S. Typhimurium (b) and of strain Lb23PP against E. coli O157:H7 (c) .

a

b

c

Mean SD Mean SD Mean SD Mean SD Mean SD VA Mean SD

Lb01PP _{10.36 0.22 -- -- -- -- -- -- 9.39 0.97 + 7.92 0.19} Lb02E _{6.68 0.64 5.20 0.54 -- -- -- -- 8.71 0.78 ++ 4.03 0.61} Lb05CA _{10.63 1.25 -- -- -- -- -- -- 12.55 0.35 + 9.87 2.04} Lb06PP _{11.74 0.18 9.17 0.43 5.90 0.78 4.73 0.09 12.64 0.35 ++ 9.01 0.23} Lb07PP _{12.47 0.42 10.77 0.81 4.78 0.43 5.03 0.18 11.24 0.24 ++ 9.66 1.49} Lb17PP _{7.45 0.23 -- -- -- -- 6.43 0.06 9.17 1.31 ++ 6.25 1.15} Lb20PP _{9.74 2.47 9.77 1.95 5.49 0.28 -- -- 7.52 0.00 + --} --Lb21PP _{6.13 0.40 -- -- 4.46 0.14 5.42 0.85 8.33 0.38 + 9.25 0.82} Lb23PP _{5.22 0.57 6.68 0.57 7.06 0.14 8.08 0.54 7.09 0.58 + 6.56 0.91}