Survey on mycoflora of cow and buffalo dairy products from Southern Italy

International Journal of Food Microbiology 69 Ž2001. 141–146 www.elsevier.comrlocaterijfoodmicro

Short communication

Survey on mycoflora of cow and buffalo dairy products from Southern Italy F. Minervini a,) , M.T. Montagna b, G. Spilotros b, L. Monaci a , M.P. Santacroce b, A. Visconti a a

b

Istituto Tossine e Micotossine da Parassiti Vegetali, CNR, Viale Einaudi 51, 70125 Bari, Italy Dipartimento di Medicina Interna e Medicina Pubblica, UniÕersita` di Bari, P.zza G. Cesare, 70124 Bari, Italy

Abstract Economic losses of dairy products due to spoilage by yeasts have been increasing in European companies because of the reduced use of preservatives, packaging in modified atmospheres, or new formulations that do not strictly control the growth of these organisms. This study reports the results of a survey of yeast species and populations in 145 samples of cow and buffalo dairy products collected in some regions of Southern Italy. Yeasts were isolated from 74% and 57% of cow and buffalo products, respectively. Candida inconspicua was the predominant species in unripened products from cow’s milk, while C. famata was detected in medium and long-term ripened dairy products, mostly in association with other yeasts and with moulds belonging to the genus Penicillium. For dairy products produced from buffalo milk, C. inconspicua was the most important yeast frequently isolated from dairy products. Total yeast populations ranged from 5 = 10 2 to 5 = 10 5 cfurg, indicating a good hygienic quality of the products. The isolation of C. albicans from one stracciatella sample is noteworthy, as this yeast represents a potential contamination by human. Even though yeasts are considered as environmental contaminants, the occurrence of some of them in dairy products at high levels could represent a risk for human health, in particular for immunocompromised patients. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Cow; Buffalo; Dairy products; Yeasts; Spoilage

1. Introduction Yeasts are known to play an important role in many cheese varieties, contributing with their metabolic properties to the ripening process. They have been used as starter cultures for soft cheese

)

Corresponding author. Tel.: q39-80-548-6073; fax: q39-80548-6063. E-mail address: [email protected] ŽF. Minervini..

production or in the maturation and aroma formation in Camembert cheese ŽJakobsen and Narvhus, 1996.. Yeasts also show a beneficial effect on the quality of dairy products because they may inhibit or eliminate undesired microorganisms and contribute to the fermentation or maturation process by supporting the function of the starter culture. Yeasts produce metabolites, e.g. short-chain fatty acids and other compounds, with known toxic effects against undesired microorganisms in the intestinal tract ŽJakobsen and Narvhus, 1996.. Yeasts can have a negative effect as spoilage organisms in fermented milk and

0168-1605r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 Ž 0 1 . 0 0 5 8 3 - 9

142

F. MinerÕini et al.r International Journal of Food Microbiology 69 (2001) 141–146

cheeses. In dairy products, they are considered among the secondary microflora because they have limited hygienic significance and can affect product shelf-life ŽRossi et al., 1997.. In recent years, economic losses due to spoilage by yeasts have increased in European companies because of less severe preservation procedures Žlower concentrations of preservatives., packaging in modified atmospheres, or new formulations that occasionally allow the growth of yeasts ŽFleet, 1990.. Many yeast contaminants can develop if good manufacturing practice is neglected, e.g. poor factory hygiene, omitted preservatives, inadequate pasteurising temperatures, and poor quality of raw materials. Recent studies have shown a possible role of yeasts, such as C. lipolytica, Exophiala spinifera, Saccharomyces telluris, and S. cereÕisiae, in the biological detoxification of mycotoxins ŽBoeswald et al., 1995; Scott et al., 1995; Karlovsky, 1999.. The mycotoxins degradation during the fermentation processes could be an attractive strategy for the safeguard of animal and human health. On the other hand, under certain circumstances, some yeasts may cause infections in man and animals, especially in patients immunocompromised or treated with bacterial antibiotics. Diseases range from superficial infections of cutaneous and mucosal sites to serious systemic disease involving the viscera and circulatory fluids ŽLaskin and Lechevalier, 1978.. The aim of this work was to evaluate the yeast populations and diversity of species found in a range of dairy products manufactured from cow’s milk and buffalo milk in some regions of Southern Italy.

2. Materials and methods The study was carried out on 145 samples of dairy products, representing 92 manufactured from cow’s milk and 53 from buffalo milk. The samples were collected from grocery stores in Apulia and Campania, two regions of Southern Italy. Samples originated from cow’s milk were distributed as follows according to the duration of ripening: Ži. 45 unripened dairy products Žno ripening required.: mozzarella Ž16., butter Ž5., fresh Apulian canestrato Ž9., burrata-stracciatella Ž9. and ricotta Ž6.;

Žii. 31 medium-ripened dairy products Žripening period: 10 days–4 months.: scamorza Ž21., gorgonzola-mascarpone Ž3. and caciotta Ž7.; Žiii. 16 long-term ripened dairy products Žripening period ) 4 months.: provolone Ž11., emmenthal Ž1. and asiago-camoscio d’oro Ž4.. The buffalo-related dairy products examined were Ži. mozzarella Ž29., Žii. ricotta Ž10., Žiii. hard ricotta Ž3., Živ. butter Ž2., Žv. ripened Ž5. and Žvi. unripened fresh cheese Ž4.. Yeast isolation was performed according to ISO 8261. In detail, 20 g of each sample were homogenised for 5 min in 180 ml peptone-water ŽDIFCOrcod. D1807-17rBecton Dickinson, Milan, Italy. by use of a Stomacher ŽPBI Internationalrcod. 19145rMilan, Italy.. Decimal dilutions in 9% Žwrv. NaCl solution ŽCarlo Erbarcod. 479687rMilan, Italy. were prepared and inoculated onto Yeast Glucose Chloramphenicol Agar ŽYGCA. medium ŽBecton Dickinsonrcod. 219001.. Plates were incubated for 3–5 days at 30 8C, after which yeast colonies were counted. The identification was carried out on five yeast colonies isolated from the last positive dilution by API20C Aux system ŽbioMerieuxrcod. ´ 20210rMarcy l’Etoil, France. and confirmed by VITEK automatic system ŽbioMerieuxrcod. 99538.. ´ The filamentous fungi were subcultured on specific media ŽPotato Dextrose Agarrcod. D0013-15; Malt Agarrcod. D0024-17; Czapek Agarrcod. D0339-17rDifcorBecton Dickinson. and identified by standard methods ŽRaper and Fennel, 1965; Schipper, 1976; Pitt, 1988; Pitt and Cruickshank, 1990..

3. Results and discussion Yeasts were isolated from 74% and 57% of samples of cow and buffalo products, respectively. 3.1. Unripened dairy products originating from cow’s milk All unripened dairy products presented a heterogeneous distribution of yeast populations. Forty-eight

F. MinerÕini et al.r International Journal of Food Microbiology 69 (2001) 141–146

yeast strains Ž63% in mixed culture and 38% in single culture. were isolated from 32 samples, giving an incidence of 71% ŽTable 1.. Burrata-stracciatella samples Žsix out of nine tested. and mozzarella samples Ž5 out of 16 tested. gave only a single yeast species, whereas mixed yeast cultures were found in fresh Apulian canestrato samples Žsix out of nine tested.. The most frequently isolated yeast was C. inconspicua Ž34% of positive samples., often in mixed culture, followed by S. cereÕisiae Ž19% of positive samples.. Populations ranged from 10 3 to 10 5 cfurg, with the fresh Apulian canestrato and butter samples giving the highest values.

143

samples, giving an incidence of 74% ŽTable 2.. C. famata was the most frequent yeast present in single culture Ž33% of total scamorza samples.; C. inconspicua was isolated from 19% of samples, mainly in mixed culture. C. zeylanoides represented 11% of total strains and it was always associated with Penicillium aurantiogriseum and P. expansum in caciotta samples and with P. roqueforti in gorgonzolamascarpone samples Žresults not shown.. Population density was 10 5 cfurg, for all tested samples. 3.3. Long-term ripened cow dairy products Sixteen yeast strains Ž50% in single culture and 50% in mixed culture. were isolated from 13 samples, giving an incidence of 81% ŽTable 3.. C. famata was the most frequently isolated yeast Ž44% of total yeasts., occurring mainly in provolone sam-

3.2. Medium-ripened cow dairy products Thirty-five yeast strains Ž74% in mixed culture and 26% in single culture. were isolated from 23

Table 1 Diversity of yeast species isolated from unripened cow dairy products Mozzarella Ž9r16. a

Burratastracciatella Ž8r9. a

Ricotta Ž2r6. a

Fresh Apuliancanestrato Ž9r9. a

Butter Ž4r5. a

Samples with one yeast species C. kefyr C. zeylanoides C. guilliermondii C. inconspicua C. famata C. maris S. cereÕisiae Rhodotorula rubra

1 – – 2 – – 2 –

1 – 1 2 – 1 1 –

– – – – – – – 1

1 1 – – 1 – – –

2 – 1 – – – – –

Samples with more than one yeast species C. inconspicua and C. tropicalis T. cutaneum and C. lusitaniae C. maris and C. inconspicua C. humicola and C. maris C. parapsilopsis and C. inconspicua C. inconspicua and C. albicans C. famataq C. guilliermondii and S. cereÕisiae C. famata and C. inconspicua C. inconspicua, C. humicola and C. zeylanoides C. famata and C. maris C. famata and C. guilliermondii S. cereÕisiae and S. rosei S. cereÕisiae and C. kefyr Mean cfurg Žpositive samples.

1 1 1 1 – – – – – – – – – 6.6 = 10 4

– – – – – 1 1 – – – – – – 5.2 = 10 4

– – – – 1 – – – – – – – – 2.2 = 10 3

1 – – – – – – 1 1 1 1 1 – 1.1 = 10 5

– – – – – – – – – – – – 1 2.7 = 10 5

a

Positive samplesrtotal analysed samples.

144

F. MinerÕini et al.r International Journal of Food Microbiology 69 (2001) 141–146

Table 2 Diversity of yeast species isolated from medium-ripened cow dairy products Scamorza Ž14r21. a

Gorgonzola-mascarpone Ž3r3. a

Caciotta Ž6r7. a

Samples with one yeast species C. parapsilopsis C. famata C. kefyr C. guilliermondii C. inconspicua

1 7 – – –

– – – – –

– – – – 1

Samples with more than one yeast species C. guilliermondiiq C. famata S. cereÕisiaeq C. maris q T. capitatum C. parapsilopsisq C. glabrata C. inconspicuaq C. glabrata C. famataq S. cereÕisiae C. zeylanoidesq moulds C. zeylanoidesq C. inconspicua C. famataq C. inconspicua C. zeylanoidesq moulds S. cereÕisiaeq C. inconspicua C. lusitaniaeq C. famata C. zeylanoidesq moulds C. maris q C. inconspicua Mean cfurg Žpositive samples.

1 1 1 1 1 – – – – – – – 1 1.6 = 10 5

1 – – – – 1 1 – – – – – – ) 5 = 10 5

– – – – – – – 1 1 1 1 1 – 2.5 = 10 5

a

Positive samplesrtotal analysed samples.

ples. Fungi belonging to genus Penicillium, Alternaria, Scopulariopsis, Aspergillus and Geotrichum were present in mixed culture Žresults not

shown.. Populations ranged from 10 2 to 10 4 cfurg, with the provolone samples giving the highest values.

Table 3 Diversity of yeast species isolated from long-term ripened cow dairy products Provolone Ž10r11. a

Emmenthal Ž1r1. a

Asiago-camoscio d’oro Ž2r4. a

Samples with one yeast species C. lipolytica C. parapsilopsis C. zeylanoides C. famata

1 1 – 4

– – – 1

– – 1 –

Samples with more than one yeast species C. famata and T. cutaneum C. maris and Scopulariopsis breÕicaulis C. famataq moulds C. zeylanoides and S. cereÕisiae C. maris, C. pelliculosa and Geotrichum penicillatum Mean cfurg Žpositive samples.

1 1 1 1 – 3.5 = 10 4

– – – – – 5 = 10 2

– – – – 1 2.8 = 10 4

a

Positive samplesrtotal analysed samples.

F. MinerÕini et al.r International Journal of Food Microbiology 69 (2001) 141–146

3.4. Products from buffalo milk Thirty-five yeast strains Ž71% in single culture and 29% in mixed culture. were isolated from 30 samples, giving an incidence of 57% ŽTable 4.. Fifty-seven percent of total yeast strains was isolated from mozzarella samples. C. inconspicua was the yeast most frequently isolated from mozzarella samples Žincidence of 41%. followed by C. kefyr Ž10%.. Populations ranged from 10 2 to 10 5 cfurg, with the mozzarella samples giving the highest values. The products prepared from cow’s milk showed a higher incidence of yeasts than those prepared from buffalo milk. However, the total yeast population in both types of products did not exceed 10 5 –10 6 cfurg. These findings are in agreement with previous reports ŽSuzzi et al., 2000. and indicate good hygienic quality, as yeast concentrations of 10 6 –10 7 cfurg develop spoilage ŽFleet, 1990.. Though some yeasts are considered as environmental contaminants, their occurrence in dairy products, especially at high levels, could also be a risk for human health, particularly in immunocompromised individuals ŽLaskin and Lechevalier, 1978; Walsh and Pizzo, 1988; Brescia et al., 1994; Schuman et al., 1998.. Yeast species potentially involved in human diseases which have

145

been found in our samples are: C. albicans, C. famata, C. guilliermondii, C. parapsilosis, C. kefyr, C. tropicalis, C. glabrata, C. inconspicua and Trichosporon cutaneum. Most dairy products from cow’s milk showed a heterogeneous distribution of yeast population. C. famata, which was found more frequently in scamorza and provolone samples, has been reported as one of the most common yeasts isolated from dairy products, together with C. sphaerica, C. kefyr, S. cereÕisiae and S. rouxii ŽJakobsen and Narvhus, 1996.. Buffalo dairy products showed C. inconspicua and C. kefyr as the predominant yeasts mainly occurring in mozzarella. A previous investigation carried out in Basilicata, another region of Southern Italy ŽSuzzi et al., 1998., show the occurrence of C. kefyr in buffalo mozzarella in agreement with our results. However C. inconspicua, the most important yeast in our study, was not found in the Basilicata survey where S. cereÕisiae, and C. sphaerica were found instead. The isolation of C. albicans from stracciatella represented a significant result because this yeast has been included among fecal indicators of human source, therefore representing a potential contamination by faeces ŽSchioppa et al., 1981; Efstratiou et al., 1998..

Table 4 Diversity of yeast species isolated from buffalo dairy products

Samples with one yeast species C. guilliermondii C. kefyr C. lusitaniae C. norÕegensis C. inconspicua C. maris C. famata S. cereÕisiae

Mozzarella Ž18r29. a

Ricotta Ž6r10. a

Butter Ž1r2. a

Ripened cheese Ž2r5. a

Unripened fresh cheese Ž2r4. a

Hard ricotta Ž1r3. a

– 3 1 – 11 – 1 –

– – 1 – 2 1 – –

– 1 – – – – –

– – 1 – – – – –

– – – 1 – – – 1

1 – – – – – – –

1 – 1 – 3.3 = 10 3

– – – – 2 = 10 5

– – 1 – 1.2 = 10 4

– – – – 4.3 = 10 4

– – – – =10 2

Samples with more than one yeast species C. inconspicuaq C. kefyr – C. maris q C. lipolytica 1 C. inconspicuaq C. lipolytica – C. inconspicuaq C. maris 1 Mean cfurg Žpositive samples. 1.9 = 10 5 a

Positive samplesrtotal analysed samples.

146

F. MinerÕini et al.r International Journal of Food Microbiology 69 (2001) 141–146

The occurrence of toxigenic fungi Ž P. commune, P. expansum, P. roqueforti, Aspergillus Õersicolor, A. sidowi, Alternaria alternata. from medium and long-term ripened dairy products is a reason for concern linked to the risk of finding mycotoxin residues in the products.

Acknowledgements This work was supported by the Project AValorizzazione dei prodotti alimentari tipici mediterraneiB Structural Funds of the European Union, Obiettivo 1-1994–1999.

References Boeswald, C., Engelhardt, G., Vogel, H., Wallnoefer, P.R., 1995. Metabolism of the Fusarium mycotoxins zearalenone and deoxynivalenol by yeast strains of technological relevance. Natural Toxins 3, 138–144. Brescia, V., Specchia, G., Montagna, M.T., Capalbo, S., Pavone, V., Tarantini, G., Palumbo, G., Pastore Citarrella, C., De Paolis, M.R., Liso, V., 1994. Incidenza delle infezioni micotiche in pazienti con neutropenia severa. Haematologica 79, 138–140. Efstratiou, M.A., Mavridou, A., Richardson, S.C., Papadakis, J.A., 1998. Correlation of bacterial indicator organisms with Salmonella spp, Staphylococcus aureus and Candida albicans in sea water. Letters in Applied Microbiology 26, 342–346. Fleet, G.H., 1990. Yeasts in dairy products. Journal of Applied Bacteriology 68, 199–211. Jakobsen, M., Narvhus, J., 1996. Yeasts and their possible beneficial and negative effects on the quality of dairy products. International Dairy Journal 6, 755–768. Karlovsky, P., 1999. Biological detoxification of fungal toxins and its use in plant breeding, feed and food production. Natural Toxins 7, 1–23. Laskin, A.I., Lechevalier, H.A., 1978. CRC Handbook of Micro-

biology. 2nd edn. Fungi, Algae, Protozoa and Viruses, vol. II, CRC Press, Florida, pp. 207–250. Pitt, J.I., 1988. A Laboratory Guide to Common Penicillium Species. 2nd edn. CSIRO, Division of Food Research, North Ryde, Australia. Pitt, J.I., Cruickshank, R.H, 1990. Speciation and synonymy in Penicillium subgenus Penicillium —towards a definitive taxonomy. In: Samson, R.A., Pitt, J.I. ŽEds.., Modern Concepts in Penicillium and Aspergillus Classification. Plenum, New York, pp. 103–118. Raper, K.B., Fennel, D.I., 1965. The Genus Aspergillus. Williams & Wilkins, Baltimore, USA. Rossi, J., Gobbetti, M., Buzzini, P., Corsetti, A., Smacchi, E., De Angelis, M., 1997. Yeasts in dairy. Annali di Microbiologia ed Enzimologia 47, 169–183. Schioppa, F., Montanaro, D., Annino, I., 1981. Candida albicans come indicatore di inquinamento in acque marine. Igiene Moderna 76, 1235–1245. Schipper, M.A.A., 1976. On Mucor circinelloides, Mucor racemosus and related species. Studies in Mycology, 12, Central Bureau Voor Achimmelcultures, Baarn, Netherlands. Schuman, P., Sobel, J.D., Ohmit, S.E., Mayer, K.H., Carpenter, C.C., Rompalo, A., Duerr, A., Smith, D.K., Warre, D., Klein, R.S., 1998. Mucosal candidal colonization and candidiasis in women with or at risk for human immunodeficiency virus infection. HIV epidemiology research study ŽHERS. group. Clinical Infectious Diseases 27, 1161–1167. Scott, P.M., Kanhere, S.R., Lawrence, G.A., Daley, E.F., Farber, J.M., 1995. Fermentation of wort containing added ochratoxin A and fumonisins B1 and B2. Food Additives and Contaminants 12, 31–40. Suzzi, G., Gardini, F., Lanorte, M.T., Caruso, M.C., Fiore, C., 1998. A survey of yeasts from water-buffalo mozzarella cheese produced in Basilicata. Proceedings of the Symposium. Yeasts in Dairy Industry: Positive and Negative Aspects, 2–3 September 1996, Copenhagen ŽDenmark. IDFSI 9801, pp. 44–49. Suzzi, G., Lombardi, A., Lanorte, M.T., Caruso, M., Andrighetto, C., Gardini, F., 2000. Phenotypic and genotypic diversity of yeasts isolated from water-buffalo Mozzarella cheese. Journal of Applied Microbiology 88, 117–123. Walsh, T., Pizzo, J.E., 1988. Nosocomial fungal infections: a classification for hospital acquired fungal infections and mycoses arising from endogenous flora or reactivation. Annual Review of Microbiology 42, 17–45.