UDK 637 ISSN 1581-9175 Acta agriculturae slovenica Letnik 84, številka 1 Volume 84, Number 1 Doslej: ZBORNIK BIOTEHNIŠKE FAKULTETE UNIVERZE V LJUBLJANI Kmetijstvo. Zootehnika Previously: RESEARCH REPORTS BIOTECHNICAL FACULTY UNIVERSITY OF LJUBLJANA Agriculture. Zootechny 84-1 Ljubljana, Acta agriculturae slovenica str. 1–90 december 2004 Acta agriculturae slovenica, 84(december 2004)1 Acta agriculturae slovenica Doslej: Zbornik Biotehniške fakultete Univerze v Ljubljani. Kmetijstvo. Zootehnika Izdaja Glavni in odgovorni urednik Tehnični urednik Uredniški odbor Jezikovni pregled Razmnoževanje Naslov uredništva E-pošta Domača stran Letna naročnina Posamezna številka Račun Sofinancira Zbornik redno selektivno zajemajo Dokumentacijska obdelava Publikacije v zameno za Zbornik pošljite na naslov Biotehniška fakulteta Univerze v Ljubljani, Jamnikarjeva 101, SI-1111 Ljubljana. Letno izhajata dva letnika vsak z dvema številkama. prof. dr. Peter DOVČ Jože STOPAR prof. dr. Tajana ČERNY (Zagreb), akad. prof. dr. Remzi BAKALLI (Athens, ZDA), prof. dr. Zdenko PUHAN (Zürich), dr. Michel BONNEAU (Saint Gilles), prof. dr.dr.h.c. Franz PIRCHNER (Innsbruck), prof. dr. Jasna M.A. STEKAR (Ljubljana), dr. Drago BABNIK (Ljubljana), prof. dr. Jernej TURK (Maribor), izr.prof. dr. Dejan ŠKORJANC (Maribor), doc. dr. Slavica GOLC TEGER (Ljubljana), izr.prof. dr. Milena KOVAČ (Ljubljana) Vanda ŠUŠTERŠIČ Tiskarna Pleško d.o.o., Rožna dolina, cesta IV/32-36, SI-1000 Ljubljana v 450 izvodih Groblje 3, SI-1230 Domžale, tel.: 01 7217 800, telefaks: 01 7241 005 peter.dovc@bfro.uni-lj.si http://www.bfro.uni-lj.si/zoo/publikacije/zbornik/ 6 000 SIT, za tujino 35 USD 4 000 SIT, za tujino 25 USD 01100-6030707410, sklic na številko 40-521-200341 Ministrstvo za šolstvo, znanost in šport Republike Slovenije AGRIS, CAB Abstracts, COBISS in FSTA Mednarodna: Slovenski nacionalni center AGRIS Domača: INDOK Oddelka za zootehniko Centralna knjižnica Biotehniške fakultete Univerze v Ljubljani, Jamnikarjeva 101, SI-1111 Ljubljana, p.p. 2995 Avtorska pravica © 2004 Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za zootehniko Acta agriculturae slovenica Previously: Research Reports Biotechnical Faculty University of Ljubljana. Agriculture. Zootechny Issued by Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1111 Ljubljana., Slovenia. Editor-in-Chief Technical Editor Editor Board Proof Reading Printed by Address of Editor E-mail Home page Annual subscription Individual issue No. of Bank Account SWIFT Code Subsides by Res. Reports are regularly indexed and abstracted by Prof. Peter DOVČ, Ph.D. Jože STOPAR Prof. Tajana ČERNY, Ph.D. (Zagreb), Academician Prof. Remzi BAKALLI, Ph.D., (Athens, ZDA), Prof. Zdenko PUHAN, Ph.D. (Zürich), Michel BONNEAU, Ph.D. (Saint Gilles), Prof. Dr.h.c. Franz PIRCHNER, Ph.D. (Innsbruck), Prof. Jasna M.A. STEKAR, Ph.D. (Ljubljana), Drago BABNIK, Ph.D. (Ljubljana), Prof. Jernej TURK, Ph.D. (Maribor), Assoc.Prof. Dejan ŠKORJANC, Ph.D. (Maribor), Ass.Prof. Slavica GOLC TEGER, Ph.D. (Ljubljana), Assoc.Prof. Milena KOVAČ, Ph.D. (Ljubljana) Vanda ŠUŠTERŠIČ Tiskarna Pleško d.o.o., Rožna dolina, cesta IV/32-36, SI-1000 Ljubljana, Slovenia, in 450 copies Groblje 3, SI-1230 Domžale, Slovenia, Tel.: +386 1 7217 800, Telefaks: +386 1 7241 005 peter.dovc@bfro.uni-lj.si http://www.bfro.uni-lj.si/zoo/publikacije/zbornik/ 6 000 SIT, for foreign countries 35 US$ 4 000 SIT, for foreign countries 25 US$ 27620-5085063007-040 DEŽELNA BANKA SLOVENIJE d.d., Kolodvorska 9, 1000 LJUBLJANA SZKB SI-2X Ministry of Education, Science and Sport of Republic Slovenia AGRIS, CAB Abstracts, COBISS and FSTA Indexing, Classification and Networking International: Slovene National AGRIS Center National: INDOC of zootechnics Please, address exchange publication to Central Library of the Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1111 Ljubljana, P.O. Box 2995, Slovenia Copyright © 2004 University of Ljubljana, Biotechnical Faculty, Zootechnical Department Acta agriculturae slovenica, 84(december 2004)1, 1-2. UDK 637 ISSN 1581-9175 Acta agriculturae slovenica Letnik 84 Ljubljana, december 2004 Številka 1 VSEBINA / CONTENTS stran page Mlekarstvo / Dairying Characterization of autochthonous lactic acid bacteria from an artisanal Italian cheese Giuseppe ZEPPA, Maria Grazia FORTINA, Paola DOLCI, Anna ACQUATI, Annibale GANDINI and Pier Luigi MANACHINI ........................................................................................ 3 Traditional manufacturing of hard chese – kachkaval on Stara Planina mountain Zora MIJAČEVIĆ and Snežana BULAJIĆ .................................................................................. 11 Use of mono- and sesquiterpenes for characterisation of mountain cheeses Giuseppe ZEPPA, Manuela GIORDANO, Marta BERTOLINO and Vincenzo GERBI ............. 17 Enterococci in cheese – phenotypization and antibiotic resistance Snežana BULAJIĆ and Zora MIJAČEVIĆ .................................................................................. 25 Enumeration, isolation, and identification of bifidobacteria from dairy products Eva VLKOVÁ, Vojtěch RADA and Iva TROJANOVÁ .............................................................. 31 Diagnostic use of proficiency testing in dairy laboratory Oto HANUŠ, Václava GENČUROVÁ, Pavel HERING, Miloš KLIMEŠ and Radoslava JEDELSKÁ ................................................................................................................................... 37 The microbiological quality of some critical control points in the cheese production of individual Slovenian cheese-makers Karmen GODIČ TORKAR and Slavica GOLC TEGER .............................................................. 43 Ekonomika / Economics The consequences of changing conditions of the European dairy sector for the strategies of dairy companies Hannes WEINDLMAIER ............................................................................................................. 63 Subject index by Agrovoc descriptors Tomaž BARTOL ........................................................................................................................... 81 Subject index by Agris category codes Nataša SIARD ............................................................................................................................... 83 Abecedno kazalo avtorjev .......................................................................................................... 85 Navodila avtorjem ....................................................................................................................... 87 Notes for authors ......................................................................................................................... 89 Acta agriculturae slovenica, 84(december 2004)1, 3-9. http://www.bfro.uni-lj.si/zoo/publikacije/zbornik Agris category codes: Q01 COBISS Code 1.01 CHARACTERIZATION OF AUTOCHTHONOUS LACTIC ACID BACTERIA FROM AN ARTISANAL ITALIAN CHEESE Giuseppe ZEPPA a), Maria Grazia FORTINA b), Paola DOLCI a), Anna ACQUATI a), Annibale GANDINI a) and Pier Luigi MANACHINI b) a) DI.VA.P.R.A., Settore Microbiologia e Industrie agrarie, Universita degli Studi di Torino, Via L. da Vinci, 44, 10095, Grugliasco, Italy. b) DI.S.T.A.M., Settore Microbiologia Industriale, Universita degli Studi di Milano, Via Celoria 2, 20133, Milano, Italy. Received June 10, 2004, accepted October 15, 2004. Delo je prispelo 10. junija 2004, sprejeto 15. oktobra 2004. ABSTRACT We studied the natural lactic bacteria population of an artisanal Italian cheese, Toma piemontese POD, from Piedmont (Northwest Italy), in order to select new strains to be used as starters in large-scale production. Isolates collected from curd and ripened artisanal cheeses were identified by the combined use of PCR 16S-23S rDNA spacer analysis, species-specific probes and 16S rDNA sequencing. Lactococci constitued 67% of the coccal isolates. Enterococci were also isolated together with strains of Streptococcus macedonicus and S. thermophilus. Lactobacilli were only detected in three samples of curds. Acidification and proteolytic activity and aroma production were also determined for each isolate. On the basis of the results a few isolates were selected and used as starters in cheesemaking trials with both raw and pasteurised milk. The produced cheeses were sensory evaluated and two of them showed typical Toma piemontese taste and flavour. The results suggest the possibility to use these new starters in both dairy industry and artisanal cheesemaking to improve product quality. Key words: milk products / autochthonous cheese / cheese Toma piemontese / microbiology / lactic acid bacteria / strains / starters / Italy KARAKTERIZACIJA MLEČNOKISLINSKIH BAKTERIJ V AVTOHTONEM KMEČKEM ITALIJANSKEM SIRU IZVLEČEK Preučevali smo naravne populacije mlečnokislinskih bakterij v avtohtonem kmečkem italijanskem siru “Toma piemontese POD” iz Piemonta v severozahodni Italiji, da bi pridobili nove seve, ki bi jih lahko uporabljali v industrijski proizvodnji. Izolate, ki smo jih osamili iz sirnine in zrelega avtohtonega sira smo identificirali s pomočjo kombinirane uporabe različnih metod in sicer analizo vmesne regije 16S-23S rDNA z verižno reakcijo s polimerazo, uporabo vrstno specifičnih začetnikov in sekvenciranjem 16S rDNA. Lactococci so predstavljali 67 % vseh izoliranih kokov. Enterokoke smo izolirali skupaj s sevi vrst Streptococcus macedonicus in S. thermophilus. Laktobacile smo odkrili samo v treh vzorcih sirnine. Za vsak izolat smo ugotavljali tudi proteolitično aktivnost, acidifikacijsko sposobnost in proizvodnjo arome. Na osnovi rezultatov smo izbrali nekatere isolate in jih kot starterske culture uporabili v poskusih izdelave sira in sicer iz surovega in pasteriziranega mleka. Sire smo senzorično ocenili in dva med njimi sta imela za “Toma piemontese» značilen okus in vonj. Naši rezultati nakazujejo možnost izboljšanja kakovosti proizvodov z uporabo teh novih starterskih kultur v sirarski industriji in v kmečki avtohtoni proizvodnji sirov. Ključne besede: mlečni izdelki / avtohtoni sir / sir Toma piemontese / mikrobiologija / mlečnokislinske bakterije / sevi / starterske kulture / Italija 4 Acta agriculturae slovenica, 84(december 2004)1. INTRODUCTION In recent years several studies have been carried out to isolate and identify autochthonous lactic bacteria from both raw milk and artisanal cheeses produced with no addition of any starter cultures (Cogan et al., 1997; Coppola et al., 2001). Increasing information on the natural microbial population present in dairy products can help to prevent the loss of microbial biodiversity in typical foods and consequently the loss of a wide range of cheeses produced by different methods whose typical features depend on local and regional traditions and on the indigenous microbial population present in raw milk and selected by the cheesemaking environment. Due to cheesemaker’s increasing demand for new strains to improve cheese quality, we isolated and identified strains from Toma piemontese POD (Protected Origin Denomination) an artisanal cheese produced in Piedmont (Northwest Italy) to be selected and used as starters in both large-scale and artisanal cheesemaking. Toma is a semi-cooked cheese which is produced in Piedmont from raw milk warmed to 37– 40 °C. Rennet is added at a concentration of 0.15–0.20 mL L–1 and the clotting time is established visually by the cheesemaker. The curd is cut into 5–10 mm particles and collected with muslin, pressed and drained for 24 h. Cheese is ripened at 6–10 °C and 85% relative humidity for 30–40 days. The production and the ripening process depend entirely on the natural microbial population present in the milk. The first objective of this work was to study the natural bacterial population present in the production of Toma piemontese cheese, while the second objective was to select new starters to be used in both artisanal and larger-scale cheesemaking. MATERIALS AND METHODS Sampling and isolation of bacteria We collected samples from 7 dairy farms in different regions of Toma piemontese POD production area; 5 curd samples on the day of the production during the summer alpine pasture and 6 cheese samples at 30–40 days of ripening were analysed. M17 agar medium was used to isolate enterococci, after incubation at 37 °C for 24–48 h and mesophilic and thermophilic cocci, after incubation at 30 °C and 37 °C for 48 h. We used MRS agar pH 5.8 to obtain lattobacilli, after incubation at 30 °C and 37 °C for 48 h. Randomly selected colonies were purified and then submitted to microscopic examination, Gram staining. The catalase test, production of gas from D-glucose, growth at 6.5% NaCl, at 10 °C and 45 °C were evaluated as well. DNA extraction Genomic DNA for PCR reactions were extracted from 100 µl of an overnight culture diluted with 300 µl of TE 1X buffer (10 mM Tris-HCl, 1 mM Na2EDTA, pH 8.0) as described by Mora et al. (2000). Identification of isolates The isolates were identified by the combined use of PCR 16S-23S rDNA spacer analysis (RSA), species-specific PCR and 16S rDNA sequencing according to the methods suggested by the following authors: Jensen et al. (1993) for RSA analysis; Ke et al. (1999) for species-specific PCR on enterococci, Cheng et al. (1997) on Enterococcus faecium, Dutka-Malen et al. (1995) on Zeppa, G. et al. Characterization of autochthonous lactic acid bacteria from an artisanal Italian cheese. 5 E. faecalis, Lick et al. (1996) on Streptococcus thermophilus, Corroler et al. (1999) on Lactococcus lactis, Zlotkin et al. (1998) on L. garvieae, Ward and Timmins (1999) on Lactobacillus casei, L. paracasei and L. rhamnosus, Berthier and Ehrlich (1998) on L. curvatus and L. sakei. A 500 bp portion of the 16S rRNA gene was sequenced for some isolates. Amplification was performed according to the protocol used by Lane (1991). PCR products were purified and sequenced using the dideoxy chain-termination principle (Sanger et al., 1977). Taxonomic identification was performed using the Ribosomal Database Project (RPD-II) (Maidak et al., 2001). Aroma analysis Aroma analysis was performed as follows: 3 g of inoculated milk for each isolate, 10 mL of internal standard (1-heptanol: 10 µg mL–1) and 28% (w/w) of NaCl were mixed and stirred at 42 °C for 40 min. Extraction was carried out at 42 °C for 20 min in head space by SPME by DVB/Carboxen/PDMS fiber (2 cm) and desorbtion at 270 °C for 4 min in splitless. The analysis was carried out by DB-WAX capillary column according to the following operation conditions: 35 °C for 5 min; 2 °C/min to 183 °C; 5 °C/min to 210 °C; 3 min at 210 °C. Mass spectra was recorded in TIC mode, ionisation voltage of 70 eV and 33–300 amu mass range. Acidification activity In order to evaluate acidification activity of the isolates, we inoculated milk at 2% for each strain and pH measures were taken for 24 h, at incubation temperature of 37 °C. Protease activity Protease activity of the isolates was observed for each strain inoculated in milk at 2% and incubated at 37 °C for 24 h, by colorimetric determination with the Hull method. Cheesemaking trials A few isolates were selected and used as starter in cheesmaking trials. Eight cheesemakings were carried out on pasteurized cow milk by using 7 different starter mixtures of lactic bacteria isolates and one commercial starter. Two cheesemakings were carried out on raw cow milk by using starter n.7 and without the addition of any starter culture. Sensory evaluation was carried out on Toma cheese obtained at 60 days ripening. RESULTS Isolates Altogether 116 coccal isolates were collected, 53 from curd samples and 63 from cheese samples (Table 1). The viable counts on M17 plates varied from 105 to 106 cfu g–1 for curd samples; higher levels were reached in cheese samples, 108 cfu g–1. Lactobacilli were only detected in three samples and their incidence was very low, 1 to 7 cfu g–1. 6 Acta agriculturae slovenica, 84(december 2004)1. Table 1. Identification of cocci from Toma samples Preglednica 1. Identifikacija kokov v siru Toma Identification Number of strains L. lactis subsp. lactis 24 L. lactis subsp. cremoris 10 L. garvieae 44 S. suis 6 S. agalactiae 2 S. dysgalactiae 2 S. macedonicus 6 S. thermophilus 3 S. uberis 1 E. faecium 9 E. durans 4 E. faecalis 2 Enterococcus spp. 4 Identification of isolates On the basis of PCR amplification of the 16S-23S rRNA spacer region (RSA) it was possible to cluster the coccal isolates in 5 groups (Fig. 1). 1 2 3 456789 Figure 1. RSA profiles of representative coccal strains of each cluster obtained. Lanes 1–4: cluster I, II, III, IV; lanes 5–8: cluster V, subgroups a, b, c, d; lane 9: DNA Ladder Mix. Slika 1. Profili regije med rRNA geni pri izbranih sevih, ki predstavljajo posamezne skupine kokov. Steze 1–4: skupine I, II, III in IV; steze 5–8: podskupine a, b, c in d; steza 9: DNA standard. Zeppa, G. et al. Characterization of autochthonous lactic acid bacteria from an artisanal Italian cheese. 7 The isolates grouped in cluster I showed typical spacer region referable to Lactococcus lactis, the cocci belonging to cluster II showed a typical spacer region referable to L. garvieae, the ones of group III to S. thermophilus, the ones of group V to genera Enterococcus. In cluster IV six isolates were grouped showing a band at 500 bp. 16S rRNA sequence analysis was necessary to identify isolates included in cluster IV, for which no indication was obtainable by RSA analysis. The analysis of the 16S rRNA sequence together with species-specific PCR was also necessary to identify some dubious isolates belonging to the other groups. Specific PCR and RSA analysis were also used to identify lactobacilli: three strains belonged to the species Lactobacillus fermentum, one to L. coryniformis, two to L. paracasei, two to L. curvatus and two to L. rhamnosus. Aroma analysis Genetically identified isolates were submitted to aroma analysis; they were chosen among the strains obtained from curd and belonging to the species L. lactis subsp. lactis, L. lactis subsp. cremoris, S. thermophilus, L. paracasei, GC-MS analysis highlighted the presence of about 100 organic compounds mainly belonging to aldehydes, alcohols, acids, of which over 40 are not present in the milk. The processing of these data by Principal Component Analysis allowed us to group these isolates in 4 sets by using the first three components (Fig. 2). A group is formed by B18 strains belonging to the species S. thermophilus. L. lactis supsp. lactis strains A4, A1, A18 and A13, A20, A6 form respectively the second and third group. The fourth group is constituted by the only L. lactis supsp. cremoris A8 strains. Milk aroma is quite different therefore it is located in a different space (M) of the score plot. Figure 2. Score plots of the first three PCA (Principal Component Analysis) factors performed on the basis of the concentration of aromatic compounds produced by lactic acid bacteria strains. Slika 2. Predstavitev vrednosti prvih treh dejavnikov (analiza glavnih komponent) na osnovi koncentracij aromatskih sestavin, ki jih proizvajajo sevi mlečnokislinskih bakterij. 8 Acta agriculturae slovenica, 84(december 2004)1. Starters The selection of strains to be used as starters in cheesemaking trials was carried out by the choice of, at least, one isolate for each PCA group. For each group was considered the strain showing quicker acidification activity in the 24 h and higher protease activity. One L. paracasei strain has been added to starters n.2, n.4, n.6 for the high acidification and proteolytic activities showed. The bacterial composition of the seven starter mixes, chosen according to the above criteria and to the results obtained, is shown in Table 2. Generally each mix contained only two strains with the exception of starter n. 7 composed of five different strains. The mix combination of two strains of L. lactis supsp. lactis has not been considered while a commercial starter, generally used for Toma piemontese POD cheesemaking, was used. Cheesemaking trials We obtained the best sensory results from n.7 in trials with both pasteurized and raw cow milk. In both cases cheese produced from starter n.7 was better in structure, taste and aroma than cheese without the addition of any starter. Table 2. Starter compositions used in cheesemaking trials Preglednica 2. Sestava starterskih kultur, ki so bile uporabljene v sirarskem poskusu Starter Isolates n.1 B18 (S. thermophilus) – A18 (L. lactis subsp. cremoris) n.2 G1 (Lb. paracasei) – A18 (L. lactis subsp. cremoris) n.3 A6 (L. lactis subsp. lactis) – B18 (S. thermophilus) n.4 A6 (L. lactis subsp. lactis) – G1 (Lb. paracasei) n.5 A8 (L. lactis subsp. lactis) – B18 (S. thermophilus) n.6 A8 (L. lactis subsp. lactis) –G1 (Lb. paracasei) A6 (L. lactis subsp. lactis) – A8 (L. lactis subsp. lactis) – n.7 A18 (L. lactis subsp. cremoris) – B18 (S. thermophilus) – G1 (Lb. paracasei) n.8 Commercial starter Toma cheese obtained by using starter n.7 in raw milk compared to the one without starter is shown in Fig. 3. The relative sensory evaluations showed that the first one is a good product characterized by typical aroma and taste, proper eyes and structure while the other one showed defective structure and strong and bitter aroma due to lipolysis and proteolysis. CONCLUSIONS The results of the present work represent the first approach to understanding the bacterial population involved in traditional Toma piemontese POD cheese. The technological performance of these strains suggest the possibility of their use in the production of Toma piemontese POD cheese in order to improve and standardize product quality. Further investigations to prove the technological characteristics of these strains and their stability will be needed. New cheesemakings are still in progress to test new starter combinations. Zeppa, G. et al. Characterization of autochthonous lactic acid bacteria from an artisanal Italian cheese. 9 --------***»-._a-i_. Toma cheese produced from raw cow milk using starter n.7 (a) and with no addition of any starter (b). Sir Toma, izdelan iz surovega mleka ob uporabi starterske kulture št. 7 (a) in brez starterske culture (b). REFERENCES Berthier, F./ Ehrlich, S.D. Rapid species identification within two groups of closely related lactobacilli using PCR primers that targeted the 16S-23S rRNA spacer region. FEMS Microbiol. Lett., 161(1998), 97–106. Cheng, S./ McCleskey, F.K./ Gress, M.J./ Petroziello, J.M./ Liu, R./ Namdari, H./ Beninga, K./ Salmen, A./ Del Vecchio, V.G.A. PCR assay for identification of Enterococcus faecium. J. Clin. Microbiol., 35(1997), 1248–1250. Cogan, T.M./ Barbosa, M./ Beuvier, E./ Bianchi-Salvadori, B./ Cocconcelli, P.S./ Fernandes, I./ Gomez, M.J./ Gomez, R./ Kalantzopoulos, G./ Ledda, A./ Medina, M./ Rea, M.C./ Rodriguez, E. Characterization of the lactic acid bacteria in artisanal dairy products. J. Dairy Res., 64(1997), 409–421. Coppola, S./ Blaiotta, G./ Ercolini, D./ Moschetti, G. Molecular evaluation of microbial diversity occurring in different types of Mozzarella cheese. J. Appl. Microbiol., 90(2001), 414–420. Corroler, D./ Desmasures, N./ Guéguen, M. Correlation between polymerase chain reaction analysis of the histidine biosynthesis operon, randomly amplified polymorphic DNA analysis and phenotypic characterization of dairy Lactococcus isolates. Appl. Micobiol. Biotechnol., 51(1999), 91–99. Dutka-Malen, S./ Evers, S./ Courvalin, P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbiol., 33(1995), 24–27. Hull, M.E. Studies on milk proteins. II Colorimetric determination of the partial hydrolysis of the proteins in milk. J. Dairy Res., 46(1947), 573–576. Jensen, M.A./ Webster, J.A./ Strauss, N. Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl. Environ. Microbiol., 59(1993), 945–952. Ke, D./ Picard, F.J./ Martineau, F./ Ménard, C./ Roy, P.H./ Ouellette, M./ Bergeron M.G. Development of a PCR assay for rapid detection of enterococci. J. Clin. Microbiol., 37(1999), 3497–3503. Lane, D.J. 16S/23S rRNA sequencing. In: Nucleic Acid Techniques in Bacterial Systematics (Eds.: Stackebrandt, E., Goodfellow, M.). New York, Wiley, 1991, 115–175. Lick, S./ Keller, M./ Bockelmann, W./ Heller, K.J. Rapid identification of Streptococcus thermophilus by primer-specific PCR amplification based on its lacZ gene. Syst. Appl. Microbiol., 19(1996), 74–77. Maidak, B.L./ Cole, J.R./ Lilburn, T.G./ Parker Jr., C.T./ Saxman, P.R./ Farris, R.J./ Garrity, G.M./ Olse, G.J./ Schmidt, T.M./ Tiedjie, J.M. The RDP-II ribosomal database project. Nucleic Acid Res., 29(2001), 173–174. Mora, D./ Parini, C./ Fortina, M.G./ Manachini, P.L. Development of molecular RAPD marker for the identification of Pediococcus acidilactici strains. Syst. Appl. Microbiol., 23(2000), 400–408. Sanger, F./ Nicklen, S./ Coulson, A.R. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74(1977), 5463–5467. Ward, L.J.H./ Timmins, M.J. Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction. Lett. Appl. Microbiol., 29(1999), 90–92. Zlotkin, A./ Eldar, A./ Ghittino, C./ Bercovier, H. Identification of Lactococcus garvieae by PCR. J. Clin. Microbiol., 36(1998), 983–985. Figure 3. Slika 3. Acta agriculturae slovenica, 84(december 2004)1, 11-15. http://www.bfro.uni-lj.si/zoo/publikacije/zbornik Agris category codes: Q01 COBISS Code 1.04 TRADITIONAL MANUFACTURING OF HARD CHESE – KACHKAVAL ON STARA PLANINA MOUNTAIN Zora MIJAČEVIĆ a) and Snežana BULAJIĆ b) a) Univ. of Belgrade, Fac. of veterinary medicine, Dept. of hygiene and technology of food products of animal origin, Bulevar JA 18, 11000 Belgrade, Serbia, Prof., Ph.D. b) Univ. of Belgrade, Fac. of veterinary medicine, Dept. of hygiene and technology of food products of animal origin, Bulevar JA 18, 11000 Belgrade, Serbia, M.Sc. Received June 10, 2004, accepted October 15, 2004. Delo je prispelo 10. junija 2004, sprejeto 15. oktobra 2004. ABSTRACT Nomads from Greece implemented the tradition of kachkaval cheese making on Stara Planina, 100 years ago. At the beginning, the kachkaval was made exclusively from sheep’s milk, during the grazing period. In the past, kachkaval was made on «bačija« dairies, and besides the kachkaval the significant by-products were ghee and «urda«. The historical data showed that in the year 1903, 160 wagons of kachkaval were exported to Vienna and Budapest. The changes in animal breeding conditions and increasing number of cattle in this region lead to significant reduction in number of sheep, and consequently caused the discontinuance in production of kachkaval. Nowadays, there are few “bačija”-dairies on Stara Planina Mountain which are producing kachkaval in the traditional way. The characteristics of chemical content of traditionally produced kachkaval are: 60.79±3.37% dry matter, 29.50±2.25% milk fat, 55.57±3.55% moisture on a fat-free basis, 48.52±2.41% fat in dry matter, 24.99±1.60% proteins, 3.8±2.57% NaCl and pH value 5.62±0.09. The traditionally made kachaval has a shape of a flatten cylinder, with the weigh of 6–7 kg, bright-yellow crust and firm dough. The traditional cheese making of kachkaval comprises the hand made process of «pasta filatta« cheese and for the end product a spicy and salty taste are typical. Revitalizing the traditional kachkaval production also means the preservation of cultural identity of the region of Stara Planina. Key words: milk products / cheese / kachkaval / Stara Planina / traditional cheese making / Serbia TRADICIONALNA IZDELAVA TRDEGA SIRA KAČKAVALJ NA STARI PLANINI IZVLEČEK Tradicija proizvodnje sira kačkavalj na Stari Planini izvira od nomadskih pastirjev iz Grčije, ki so jo tja prinesli pred več kot 100 leti. Na začetku so kačkavalj izdelovali izključno iz ovčjega mleka v pašnem obdobju. Kačkavalj so izdelovali v mlekarnah – “bačijah”, poleg sira pa sta bila tipična stranska proizvoda še malso in “urda”. Po zgodovinskih podatkih so leta 1903 izvozili 160 vagonov kačkavalja s Stare Planine na Dunaj in v Budimpešto. S spremembo pogojev v živinoreji in s povečanjem staleža govedi, se je število ovac na Stari Planini bistveno zmanjšalo, zato je tudi proizvodnja kačkavalja skoraj zamrla. Danes še obstoja nekaj mlekarn –“bačij” na področju Stare Planine, ki še vedno proizvajajo kačkavalj po tradicionalnem postopku. Kačkavalj, izdelan na tradicionalen način, vsebuje 60,79±3,37 % suhe snovi, 29,50±2,25 % maščobe, 48,52±2,41 % maščobe v suhi snovi, 24,99±1,60 % beljakovin, 3,8±2,57 % NaCl, in ima pH vrednost 5,62±0,09. Tradicionalna oblika kačkavalja je sploščen cilinder, ki tehta 6–7 kilogramov, ima svetlo rumeno skorjo, in čvrsto testo. Tradicionalna izdelava kačkavalja temelji na ročni pripravi pasta filata sira (kuhan sir), za končni izdelek pa je značilen pikanten in slan 12 Acta agriculturae slovenica, 84(december 2004)1. okus. Oživljanje tradicije izdelave kačkavalja na področju Stare Planine je pomembno tudi zaradi ohranjanja kulturne identitete te regije. Ključne besede: mlečni izdelki / sir / kačkavalj / Stara Planina / tradicionalna izdelava sira / Srbija THE HISTORY Hard cheese, so called kachkaval, is produced in Mediterranean region, including Adriatic and Black Sea regions, in other words territory once under the rule of Ancient Greece, Roman Empire and their eastern and southern colonies. Based on written historical monuments, we can conclude that kachkaval has a very long history. Columella, the roman writer (Pejić, 1956) from 68 AD described the process of cheese making »manum pressum«, which is almost identical to the production of kachkaval. The region where kachkaval was manufactured in the past and where it is manufactured today is in the fact the region where during the second century BC and all up to the seventh century of the new era great migrations of nomadic tribes have occurred. Their major agricultural activity was cattle and sheep breeding. On the Balkan Peninsula, especially in Romania, Bulgaria, Greece and Serbia, the animal breeding was mostly in the hands of Tzintzars, who were manufacturing predominantly hard cheese – kachkaval. According to historical data Tzintzars were natives of Balkan Peninsula, who settled there before Romans, and even perhaps before the ancient Greeks. According to the historical material (Pejić, 1956), it can be most certainly presumed that the manufacturing of cheese was brought to the Balkan Peninsula by nomadic tribes from the East. Later, the manufacturing of this cheese was brought to Italy and from there to Britain during the period of Julius Caesar. In Britain, the technology of making cheese has been adapted to climatic conditions and leading to the development of the technology for Cheddar cheese production. This ancient technology of cheese making is mostly spread on the Balkan Peninsula and in Italy, as well as in the southern regions of Russia (the Crimea, Be Arabia, South Ukraine), in Turkey, Algeria, Tunisia, Egypt and Morocco, that is in the regions with warm and dry climate, hilly relief and developed sheep breeding (Pejić, 1956). In each of these countries with developed manufacturing of hard cheese during the time some varieties or types of hard cheese developed distinguishing themselves according to the properties and quality, such as hard cheese from Šarplanina Mountain, Pirot hard cheese in Serbia, Pirdop cheese from Bulgaria, Penlu from Romania and Tesalia and Epiria cheese from Greece. The origin of the name kachkaval can be found in the language of the people who most probably brought kachkaval to the Balkan Peninsula from where it spread further. In the Tzintzar language the word ýkačý means cheese. The story about hard cheese production on Stara Planina Mountain necessarily includes the nomadic sheep breeders, who are known as ýCrnovunciý (Blackwool people). They were named after the black colour of the wool of their sheep. In the period from the end of the 19th century up to the third decade of the 20th century “Crnovunci” populated the pastures of Stara Planina Mountain with their flocks of 500–1000 sheep. Large quantities of sheep milk, which gave their flocks, “Crnovunci” processed into white cheese and kept it in bellows. In order to prolong the durability of this cheese, it was soaked into hot water, mixed and salted, that is, in other words, the processing of milk into hard cheese – kachkaval had started. The skill of making kachkaval “Crnovunci” have passed on to the people of Dojkinci village, who improved it to the perfection and kept until today (Petrović, 1997). Production of kachkaval on Stara Planina Mountain is connected with small dairies called «bačija». «Bačija» dairy represents cooperative organization for joint sheep keeping, pasture, production and processing of all products – wool, meat and milk. Peasants realized that small Mijačević, Z. and Bulajić, S. Traditional manufacturing of hard chese – kachkaval on Stara Planina mountain. 13 number of sheep is of no use, therefore they associated in all activities relating to sheep breeding and production at dairies – «bačije» (Stojanović et Katić, 2003). Manufacturing of hard cheese – kachkaval and other products was the same at all “bačija” dairies on Stara Planina Mountain. It is known that they were made from light material that could be transported, premises were exposed to draft, and their size varied depending on the quantity of milk. Processing of milk was carried out in wooden tubs containing warm water where the «baskia» was fumed, and also dairies – «bačija» had tables for casting and salting as well as a table for cheese ripening (Pejić, 1956). PRODUCTION STAGES IN MANUFACTURING OF HARD CHEESE – KACHKAVAL IN DAIRY PLANT IN THE VILLAGE OF DOJKINCI ON STARA PLANINA MOUNTAIN On Stara Planina Mountain, in the village of Dojkinci, dairy plant has a daily capacity for processing of 2000 l of milk into kachkaval. Altogether there are 7 similar dairies on Stara Planina Mountain at present that are processing larger or smaller quantities of milk. Milk is combined sheep’s and cow’s milk, although according to the original technology of kachkaval production only sheep milk is to be used. Prepared quantity of milk (800 l) from Dojkinci is heated until the temperature of 65–70 °C is achieved. This temperature is achieved in approximately two hours and subsequently warmed milk is mixed with remaining milk coming from other villages. Temperature of mixed milk is 30 °C and acidity 8–8.2 °SH. The curdling temperature does not change during the whole phase of curdling which lasts for 60 minutes. The phase of processing curd starts by curd chopping up by means of the cross and lasts for 5 minutes, the temperature of separated whey is 29.6 °C and acidity 6.6oSH, while the temperature of the curd is 29 °C and acidity 5.2 °SH. After separating a part of whey, the curd is again intensively mixed to separate the grains and again a part of whey is being separated, with acidity of 6.4 °SH, and temperature 29.9 °C, while the curd has acidity of 10.8 °SH and temperature of 29.5 °C. After that the warm water from the boiler is added and the temperature of curd is lifted to 34.9 °C. The duration of the phase of drying the curd is 10 minutes, until the required strength of the grains has been reached. The acidity of whey is 9.6 °SH. The curd is then left in the bath, pressed for 10 min to separate whey and then cut, taken out from the bath and transferred to the drainer on to the cheese-making table, grained, allotted and pressed for 20 minutes, the pressing being achieved by screwing the press according to the experience. After pressing the lump is cut into pieces, taken out from the drainer and left on the draining table until the next day. The temperature of the room is 20.7 °C, and the period of ripening of baskia is 19–20 hours. The room temperature during the night is 15–16 °C. After the acidity has been inspected by spreading (Fig. 1), “baskia” is chopped on the cutter like that for cabbage and steamed in the water whose temperature is 75 °C. Steaming is performed in weaved baskets (Fig. 2), containing about 6 kg of baskia, devaporated and put onto the cheese-making table (Fig. 3), salted (Fig. 4), formed into the ball (Fig. 5) and put into casts (Fig. 6). The cheese remains in the casts until the next day. Than it is taken out, weighed and transferred into the room at 20 °C for drying. After drying for 1–2 days, it is transferred into the room for cheese ripening. The temperature in the ripening room 20 °C, and the cakes of cheese are put one onto the other and salted with dry salt. Finally, the ripened kachkaval is packed into vacuum bags. Major characteristics of chemical composition of hard cheese from Stara Planina Mountain are shown in Table 1. Chemical composition of the kachkaval from Stara Planina Mountain varied and ratio between the minimum and maximum value of dry matter was 10%. It must also be pointed out 14 Acta agriculturae slovenica, 84(december 2004)1. that percentage of salt in certain kachkavals also varied and that there were samples of cheese with over 7% of salt. Table 1. Chemical composition of kachkaval from Stara Planina Mountain (Mijačević et al., 2003) Preglednica 1. Kemična sestava sira kačkavalj s Stare Planine (Mijačević in sod., 2003) Parameters / Parametri n X±s min max % dry matter % suhe snovi 7 60.79±3.37 54.55 64.05 % fat % maščobe 7 29.50±2.25 25.00 32.50 % water without fat % vode brez maščobe 7 55.57±3.55 50.07 60.60 % fat in dry matter % maščobe v suhi snovi 7 48.52±2.41 45.83 53.03 % protein % beljakovin 7 24.99±1.60 25.07 27.38 % dissolved protein % raztopljenih beljakovin 7 5.72±0.22 5.42 6.02 % NaCl 7 3.80±2.57 1.66 7.67 pH 7 5.62±0.09 5.50 5.75 oSH 7 37.82±8.41 23.20 51.20 Table 2. Sensory evaluation of cheese in different ripening stages Preglednica 2. Senzorična ocena sira v različnih fazah zorenja Ripening period of cheese Čas zorenja sira Crust Skorja Cross section Prerez Consistency Konzistenca Smell Vonj Taste Okus 2 days 2 dneva Characteristic for young cheese Technological holes, grain of not devaporated baskia, layers well expressed Soft, elastic Smells like milk Bitter 30 days 30 dni Yellow crust, integrity preserved Technological holes, layers preserved Firm Smell of sheep and cow milk Salty, bitter, fine melting in the mouth 60 days 60 dni Yellow crust, integrity preserved Technological holes Firm Smell of sheep and cow milk Salty, bitter, fine melting in the mouth Mijačević, Z. and Bulajić, S. Traditional manufacturing of hard chese – kachkaval on Stara Planina mountain. 15 THE ANALYSIS OF SENSORY CHARACTERISTICS OF KACHKAVAL FROM STARA PLANINA MOUNTAIN Sensory evaluation of 2 days old cheese in drying process, cheese that ripened for 30 and 60 days, respectively, was carried out by three evaluators. Sensory evaluation of investigated cheese is presented in Table 2. Sensory evaluation indicates that 30 days old cheese can be placed on the market labeled as cheese, ripe for consumption. However, true ripeness is achieved after the period of 60 days. In general, cheese is salty, and the cross section shows technological holes. Occurrence of technological holes is characteristic for manual processing of steamed dough, however, number of holes should be low and their size relatively small. Analysis of cheese, ripe for consumption and ripe cheese has revealed the absence of coliform bacteria and E. coli. However, some useful microorganisms were isolated that are responsible for specific taste of the cheese. In the cheese, originating from Stara Planina Mountain, the most often isolated bacteria are Enterococcus spp, Lactococcus spp., Leuconostoc spp and Lactobacilus spp., with specific characteristic being able to grow in conditions of high and low temperatures and in the broth with increased quantity of salt. CONCLUSION Since there is preserved tradition of manufacturing hard cheese – kachkaval on Stara Planina Mountain, with already famous Senokos, Dojkinci, and Pirot kachkaval, this is also the justification of the necessity for protection of kachkval produced in this region with certain label defining geographical origin of the product. Preservation of the tradition of cheese manufacturing means at the same time also preservation of the cultural identity of the region (Dozet et al., 1996, Mijačević et al., 2003a and 2003b) REFERENCES Dozet, N./ Adžić, N./ Stanišić, M./ Živić, N. Autohtoni mlječni proizvodi. Poljoprivredni institut –Podgorica, Silmir- Beograd, 1996. Mijačević, Z./ Bulajić, S. Proizvodnja sira sa geografskim obeležjima u zanatskim mlekarama. Savremena poljoprivreda, 51(2002)3–4, 379–384. Mijačević, Z./ Bulajić, S./ Nedić, D. Sir sa oznakom geografskog porekla-trend, markentiški izazov ili neminovnost. Veterinarski žurnal Republike Srpske, 3(2003a)1–2, 58–61. Mijačević, Z./ Bulajić, S./ Božić T. Tehnološke karakteristike bakterija mlečne kiseline i enterokoka izolovanih iz kačkavalja poreklom sa Stare planine. Biotechnology in animal husbandry, 19(2003b)5–6, 159–165. Pejić, O. Mlekarstvo, B Specijalni deo-sirevi. Naučna knjiga, Beograd, 1956. Petrović, M. Dojkinci. Biblioteka “Hronika sela”, Beograd, 1997. Stojanović, L./ Katić V. Istorijski značaj i proizvodnja Pirotskog kačkavalja. Mlekarstvo, (2003)13, 390–394. Acta agriculturae slovenica, 84(december 2004)1, 17-23. http://www.bfro.uni-lj.si/zoo/publikacije/zbornik Agris category codes: Q01 COBISS Code 1.01 USE OF MONO- AND SESQUITERPENES FOR CHARACTERISATION OF MOUNTAIN CHEESES Giuseppe ZEPPA a), Manuela GIORDANO a), Marta BERTOLINO a) and Vincenzo GERBI a) a) University of Turin, Department of Exploitation and Protection of the Agricultural and Forestry Resources – Laboratory of Food Technology, Via L. da Vinci 44, 10095, Grugliasco (TO), Italy. Received June 10, 2004, accepted October 15, 2004. Delo je prispelo 10. junija 2004, sprejeto 15. oktobra 2004. ABSTRACT Terpenes are volatile compounds originating exclusively from plants that can be found in dairy products. Several studies have therefore been conducted to use the terpenes to trace the geographical origin or the nature of the feed supplied to animals. The vegetation of mountain pastures varies which results in different types of foraging. Our aim was to determine if the terpene profile can be used to distinguish the mountain pasture of origin. The mono- and the sesquiterpenes of ricotta cheese samples produced during the summer in three mountain farms were then determined with SPME-GC-MS. The ricotta cheeses were produced with cow and goat milk and every three days of production a sample was analysed. Obtained results showed that these compounds vary widely between samples due to the stage of plant development but discrimination among the farms was still possible. Hence mono- and sesquiterpenes can be used not only to distinguish between summer pasture cheeses and the ones produced during other periods but also as markers of each mountain pasture. Key words: milk products / cheese / ricotta / characterisation / terpenes / sesquiterpenes / markers UPORABA MONO- IN SESKVITERPENOV ZA KARAKTERIZACIJO SIROV S PLANIN IZVLEČEK Terpeni so hlapne snovi izključno rastlinskega izvora, ki jih lahko najdemo v mlečnih izdelkih. Opravljene so bile študije, v katerih so terpene uporabljali za sledenje in ugotavljanje geografskega porekla krme, ki so jo zaužile živali. Razlike v vegetaciji na planinskih pašnikih se odražajo tudi v krmi. V tej raziskavi smo želeli proučiti možnost, če na osnovi profila terpenov lahko ugotovimo ali so se krave pasle na planinskem pašniku. Poleti smo s SPME-GC-MS ugotavljali mono- in seskviterpene v vzorcih sira “ricotta” s treh planinskih farm.. Vzorce sira “ricotta”, ki so ga izdelovali iz kravjega in ovčjega mleka smo zbirali vsak tretji dan in opravili analize. Čeprav rezultati kažejo, da se vsebnost proučevanih snovi v vzorcih, zaradi razlik v razvojni fazi rastlin, zelo razlikuje, smo lahko ločevali vzorce z različnih farm. Mono- in seskviterpene lahko uporabimo za ločevanja med siri, ki so bili izdelani med pašno sezono in izven nje pa tudi kot markerje za posamezne planinske pašnike. Ključne besede: mlečni izdelki / sir / ricotta / karakterizacija / terpeni / seskviterpeni / markerji INTRODUCTION Animal feeding is a very important factor in cheese characterisation due to its action on bacteria and milk compounds such as fats, proteins, flavours and so on. Several studies have 18 Acta agriculturae slovenica, 84(december 2004)1. highlighted the possibility to define a relation between cheese and its production area with the study of animal feed and above all with the study of terpene compounds (Dumont and Adda, 1978; Moio et al., 1996; Mariaca et al., 1997; Schehovic et al., 1998; Viallon et al., 1999; Bugaud et al., 2000; Viallon et al., 2000; Bugaud et al., 2001a; Bugaud et al., 2001b). These molecules are secondary metabolites of plants, well-known for their disinfectant (medicinal) and odorant (spice) properties. In the plant kingdom their qualitative and quantitative distribution is highly variable, but they are specie-specific thus many researchers have suggested that the analysis of such substances might improve the traceability of dairy products or meats originating from animals raised in specific geographical areas. Results have shown that the milk and the cheese from different production sites (lowland vs. highland) and seasons (winter vs. summer) can be distinguished (Jeangros et al., 1997; Bosset et al., 1999; Buchin et al., 1999; Cornu et al., 1999; Buchin et al., 2002). In fact terpene compounds are more abundant in dicotyledones than in monocothyledons which are more abundant in alpine pastures. Terpenes are also more abundant in fresh grass than in hay. The aim of this work was to verify if mono- and sesquiterpens can also be used to distinguish the products from different highlands pastures and to define a traceability system for a typical mountain cheese. MATERIALS AND METHODS Three mountain farms (Alpe Bancet, Alpe Gianna, Alpe Pra) were used in this study. The farms are located in Pellice Valley near Torino (Piedmont, North West Italy) at 1000–2500 m. A sample of ricotta cheese has been taken every four days for the period July 12/August 23 for Alpe Pra, for the period July 12/September 16 for Alpe Bancet and for the period July 14/September 16 for the Alpe Gianna. These differences of sampling are due to the different time of mountain grazing. Each sample was vacuum packed in polyethylene bags and stored to –20 °C. Samples of 2.5 g of ricotta cheese were taken and placed in a 10 mL glass vial (38 mm high and 22 mm in diameter) and sealed with 20 mm PTFE/Silicone septum caps (Supelco, Bellefonte,USA). For the conditioning, the sample vial was placed in a 53 °C water bath for 10 min. The fiber used for the extraction was a DVB/Carboxen/PDMS 2 cm stable flex fiber (Supelco, Bellefonte, USA). The volatile components were extracted using the static headspace method. During this step the fiber was exposed for 60 min in the headspace of the cheese with the vial maintained at 53 °C in a thermostatic bath. Every sample was analysed in triplicate. The adsorbed molecules were desorbed by introducing the SPME fiber into the injector of a gaschromatograph (GC17A, Shimadzu, Tokyo, Japan) at 270 °C for 6 min in splitless mode. The volatile components were separated on a DB-WAX capillary column (30 m × 0.25 mm ID; film thickness 0.25 µm; J&W Scientific Inc., Folsom, CA, USA). The oven temperature program and the operating conditions were as follow: carrier gas helium at 1 mL min–1; the column was maintained at 35 °C for 5 min, ramped at 2 °C min–1 to 173 °C, maintained at 173 °C for 1 min, ramped at 15 °C min–1 to 210 and finally maintained at 210 °C for 5 min. Mass spectra were recorded in the electron impact mode at an ionisation voltage of 70 eV in the 33–300 amu mass range. The ion source and the interface were maintained at 220 °C. Compounds identification was carried out with the mass spectra and retention times of standard compound, when available, or the NIST 12 and NIST62 mass spectral data base. Statistical analysis was performed with Statistica ver. 6.0 (Statsoft Inc., Tulsa, OK, USA). Zeppa, G. et al. Use of mono- and sesquiterpenes for characterisation of mountain cheeses. 19 RESULTS The analysis of volatile compounds highlighted the presence of twenty-two monoterpenes (over all ?-pinene, ß-pinene, camphene, p-cymene, ß-myrcene and limonene) and sixteen sesquiterpenes such as ?-caryophyllene, ?-copaene and 9-epi-caryophyllene. Twenty-one monoterpenes were detected in the Alpe Bancet ricotta, 16 in the Alpe Gianna ricotta and 20 in the Alpe Pra ricotta. In these products also 13, 11 and 16 sesquiterpenes, respectively, were detected (Table 1). Table 1. Monoterpenes (t) and sesquiterpenes (s) detected in the ricotta cheeses analysed for each mountain farm (n.i. – not identified) Preglednica 1. monoterpeni (t) in seskviterpeni (s), odkriti v siru “ricotta” s posameznih gorskih farm (n.i. – ni identificirano) t t t t t t t t t t t t t t t t t t t t t t BANCET GIANNA PRA * * * * * * * * * * * * * * * * * * * * * s s s s s s s s s s s s s s s s BANCET GIANNA PRA a-phellandrene * * 9-epi-caryophyllene * * * a-pinene * * a-caryophyllene (a-humulene) * * * a-terpineol * * a-copaene * * * ß-myrcene * a-isocomene * bornyl acetate * ß-maaliene * ß-pinene * * epi-cedrane * * * Camphene * * isocaryophyllene * * 5-3-carene * * selinene * * * dihydro carveol acetate sesquiterpene (n.i.) * * * D-verbenone * sesquiterpene (n.i.) * * Y-terpinene * * sesquiterpene (n.i.) * * * Limonene * * sesquiterpene (n.i.) * * * Linalool * * sesquiterpene (n.i.) * * * Myrtenol * * sesquiterpene (n.i.) * * p-cymene * * sesquiterpene (n.i.) * Sabinene * * valencene * * * terpene (n.i.) * * terpene (n.i.) * * terpene (n.i.) * terpene (n.i.) * * terpene (n.i.) * * terpene (n.i.) * 20 Acta agriculturae slovenica, 84(december 2004)1. Figure 1. Trend of some terpenes (reported as area of base peak ion) for the three mountain farms ricotta cheeses during the grazing. Slika 1. Trend gibanja posameznih terpenov v vzorcih sira “ricotta” s treh planinskih farm v pašni sezoni. Zeppa, G. et al. Use of mono- and sesquiterpenes for characterisation of mountain cheeses. 21 All these compounds showed a wide concentration variability due to the plant stage development and the pastured area during the mountain grazing. In Fig. 1 this effect is reported for some mono- and sesquiterpenes. 1.E+06 1.E+06 8.E+05 6.E+05 4.E+05 2.E+05 O.E+00 sabinene ;>++< :>++< ;>++< ;>++< ;>++< ;>++< ;>++< ;>++< ;>++< :>++< ;>++< ;>++< ;>++< ;>++< HPra Gianna D Bancet +++ +++ +++ +++ +++ limonene 9-epi-caryophyllene a-caryophyllene Figure 2. Area of base peak ion of two monoterpenes and two sesquiterpenes for the three mountain farms. Slika 2. Površina vrhov dveh monoterpenov in dveh seskviterpenov na treh planinskih farmah. ,- 0 4 3 2 1 0 -1 i- -2 -3 p p % p P B P ° B B p p 0 g p rB g g g g 8 8 8 ' g ai q S " S g L p p p bob 8 °o ^ 3 b b o b SJ 0 ^ % o o ° b° bo ° b -4 - 8 -6 -4 -2 0 2 46 Factor 1 Figure 3. Score-plot with the ricotta cheese samples of the three mountain farms (g - Alpe Gianna; b - Alpe Bancet; p - Alpe Pra) calculated by terpene concentrations. Slika 3. Porazdelitev vzorcev sira “ricotta” s treh planinskih farm (g - Alpe Gianna; b - Alpe Bancet; p - Alpe Pra) na osnovi koncentracije terpenov. 22 Acta agriculturae slovenica, 84(december 2004)1. Although the concentrations of these compounds vary during the pasturing season, there are high differences between the three mountain farm ricotta cheeses (Fig. 2). Mono- and sesquiterpenes are particularly abundant in the Alpe Pra products and this is due to the mountain pastures with a small percentage of graminacee and the presence of goat and ewe flocks. Concentration of mono- and sesquiterpenes in Alpe Bancet and Alpe Gianna is generally lower for the presence of cow herds and a high percentage of graminacee in the pasture. The differences between the three mountain farms can be highlighted with the Linear Discriminant Analysis calculated by areas of base peak ions for each detected terpenes (Fig. 3). The discrimination of Alpe Pra samples is excellent while for the other two farms some samples are misclassified. The refiling percentage is approximately 99% for Alpe Pra samples and 95% for the other farms. The differences between Alpe Bancet and Alpe Gianna are more evident at the end of the pasturing period (September) when, due to its northern exposure, there is still fresh grass in Alpe Gianna while the pasture is very reduced in Alpe Bancet. CONCLUSIONS This study has confirmed the presence of mono- and sesquiterpenes in the mountain ricotta cheese in the mountain ricotta cheese much like those highlighted for other cheeses. These compounds can be used not only to distinguish between summer pasture products and the ones produced during other periods but also as markers of each mountain pasture. The study has also confirmed that SPME-GC-MS is a simple and effective technique for the study of terpene compounds. ACKNOWLEDGMENTS Research supported by the Assessorato all’Agricoltura of Provincia di Torino, Italy. REFERENCES Bosset, J.O./ Jeangros, B./ Berger, T./ Bütikofer, U./ Collomb, M./ Gauch, R./ Lavanchy, P./ Scehovic, J./ Troxler, J./ Sieber, R. Comparaison de fromages a pate dure de type Gruyere produits en régions de montagne et de plaine. Rev. Suisse Agric. 31(1999), 17–22. Buchin, S./ Martin, B./ Dupont, D./ Bornard, A./ Achilleos, C. Influence of the composition of Alpine pasture on the chemical, rheological and sensory properties of cheese. J. Dairy Res. 66(1999), 579–588. Buchin, S./ Salmon, J.C./ Carnat, A.P./ Berger, T./ Bugaud, C./ Bosset, J.O. Identification de composés monoterpéniques, sesquiterpéniques et benzéniques dans un lait d’alpage tres riche en ces substances. Mitt. Lebensm. Hyg. 93(2002), 199–216. Bugaud, C./ Bornard, A./ Hauwuy, A./ Martin, B./ Salmon, J.C./ Tessier, L./ Buchin, S. Relation entre la composition botanique de vegetations de montagne et leur composition en composés volatils. Fourrages 162(2000), 141–155. Bugaud, C./ Buchin, S./ Coulon, J.B./ Hauwuy, A. Relationships between flavour and chemical composition of Abondance cheese derived from different types of pastures. Lait 81(2001a), 757–773. Bugaud, C./ Buchin, S./ Coulon, J.B./ Hauwuy, A./ Dupont, D. Influence of the nature of alpine pastures on plasmin activity, fatty acid and volatile compound composition of milk. Lait 81(2001b), 401–414. Cornu, A./ Carnat, A.P./ Martin, B./ Coulon, J.B./ Lamaisson, J.L./ Berdagué, J.L. Solid-Phase Microextraction of volatile components from natural grassland plants. J. Agric. Food Chem. 49(1999), 203–209. Dumont, J.P./ Adda, J. Occurrence of sesquiterpenes in mountain cheese volatiles. J. Agric. Food Chem. 26(1978), 364–367. Jeangros, B./ Troxler, J./ Conod, D./ Schehovic, J./ Bosset, J.O./ Bütikofer, U./ Gauch, R./ Mariaca, R./ Pauchard, J.P./ Sieber, R. Etude des relations entre les caractéristiques des erbages et celles du lait, de la creme et du fromage de type L’Etivaz ou Gruyere. Revue Suisse Agric. 29(1997), 23–34. Zeppa, G. et al. Use of mono- and sesquiterpenes for characterisation of mountain cheeses. 23 Mariaca, R.G./ Berger, T.F.H./ Gauch, R./ Imhof, M.I./ Jeangros, B./ Bosset, J.O. Occurrence of volatile mono- and sesquiterpenoids in highland and lowland plant species as possible precursors for flavour compounds in milk and dairy products. J. Agric. Food Chem. 45(1997), 4423–4434. Moio, L./ Rillo, L./ Ledda, A./ Addeo, F. Odorous constituents of ovine milk in relationship to diet. J. Dairy Sci. 79(1996), 1322–1331. Schehovic, J./ Jeangros, B./ Troxler, J./ Bosset, J.O. Effets de la composition botanique des herbages pâturés sue quelques composants des fromages de type L’Etivaz ou Gruyere. Revue Suisse Agric. 30(1998), 167–171. Viallon, C./ Martin, B./ Verdier-Metz, I./ Pradel, P./ Garel, J.P./ Coulon, J.B./ Berdagué, J.L. Transfer of monoterpenes and sesquiterpenes from forages into milk fat. Lait, 80(2000), 635–641. Viallon, C./ Verdier-Metz, I./ Denoyer, C./ Pradel, P./ Coulon, J.B./ Berdagué, J.L. Desorbed terpenes and sesquiterpenes from forages and cheeses. J. Dairy Res. 66(1999), 319–326. Acta agriculturae slovenica, 84(december 2004)1, 25-30. http://www.bfro.uni-lj.si/zoo/publikacije/zbornik Agris category codes: Q01 COBISS Code 1.01 ENTEROCOCCI IN CHEESE – PHENOTYPIZATION AND ANTIBIOTIC RESISTANCE Snežana BULAJIĆ a) and Zora MIJAČEVIĆ b) a) Univ. of Belgrade, Fac. of veterinary medicine, Dept. of hygiene and technology of food products of animal origin, Bulevar JA 18, 11000 Belgrade, Serbia, M.Sc. b) Same address as a), Prof., Ph.D. Received June 10, 2004, accepted October 15, 2004. Delo je prispelo 10. junija 2004, sprejeto 15. oktobra 2004. ABSTRACT The material of investigation was consisted of samples of fresh and ripened cheeses made from raw and cooked milk subjected to rennet or acid coagulation. The primary isolation of enterococci was carried out on kanamycin-aesculine-azide agar at 37 °C and 42 °C during 24 hours. It was isolated totally 42 strains of enterococci. The examination of antibiotic resistance/sensitivity profiles was performed by applying disk-diffusion procedure on Muller-Hinton agar. The number of enterococci determined in cheese samples depended on applied technological process. In the samples of fresh, by rennet coagulated cheese made from raw and cooked milk the number of enterococci was ranged from 8.0×104 to 9.0×106 cfu g–1, and from 4.0×107 to 4.4×107 cfu g–1, respectively. In the case of ripened cheeses made from raw and cooked milk subjected to acid coagulation the number of enterococci was ranged from <102 to 5.0×104 cfu g–1 and from 2.5×104 to 1.5×105 cfu g–1, respectively. In the samples of fresh cheeses made from raw and cooked milk subjected to acid coagulation the number of enterococci was ranged from 2.4×105 to 2.12×107 cfu-g, and from 5×104 to 6×104 cfu g–1, respectively. The phenotypic identification of isolated enterococcal strains was performed according to the following biochemical-physiological characteristics: microscopic examination (cell morphology), catalase activity, growth in MRS broth at 10 °C and 45 °C, growth at pH 9.6, growth in broth containing 6.5% NaCl, growth in 0.1% methylen blue milk, resistance at 60 °C/15 and 30 minute, Voges/Proskauer reaction, fermentation of ribose. The isolated strains of enterococci were resistant to following antibiotics: penicillin (65.82%), tetracycline (62.02%), lincomycin (68.35%), gentamycin (27.84%), neomycin (31.64%), erythromycin (31.64%) and chloramphenicol (65.82%). Key words: milk products / cheese / microbiology / enterococci / phenotypization / antibiotics / resistance ENTEROKOKI V SIRIH – FENOTOPIZACIJA IN REZISTENCA PROTI ANTIBIOTIKOM IZVLEČEK V raziskavi smo proučevali vzorce svežih in zorenih sirov, izdelanih iz surovega ali toplotno obdelanega mleka s potopkom encimske ali kislinske koagulacije. Začetno izolacijo enterokokov smo opravili na kanamicin-eskulin-azidnem agarju pri temperaturi 37 °C in 42 °C in 24-urni inkubaciji. Izolirali smo 42 sevov enterokokov. Za ugotavljanje rezistence oziroma občutljivosti izoliranih sevov proti antibiotikom smo uporabili difuzni test z diski, na Muller-Huntonovem agarju. Število enterokokov, ki so jih ugotovili v vzorcih sirov, je bilo odvisno od tehnološkega postopka izdelave sira. V svežih sirih, narejenih z encimsko koagulacijo surovega in toplotno obdelanega mleka, se je število enterokokov gibalo med 8,0×104 in 9,0×106 cfu g–1 ter 4,0×107 in 4,4×107 cfu g–1. Pri zorenih sirih, narejenih s kislinsko koagulacijo, se je število enterokovov 26 Acta agriculturae slovenica, 84(december 2004)1. gibalo od < 102–5,0×104 cfu g–1 v sirih iz surovega mleka in od 2,5×104 do 1,5×105 cfu g–1 v sirih iz toplotno obdelanega mleka. Sveži siri, narejeni s kislinsko koagulacijo surovega in toplotno obdelanega mleka so vsebovali med 2,4×105 in 2,12×107 cfu g–1, ter med 5×104 in 6×104 cfu g–1. Fenotipsko smo izolirane seve opisali na osnovi morfologije celic, katalaznega testa, rasti pri 10 °C in 45 °C, rasti v mleku z 0,1 % metilenskega modrila in pri pH 9,6, rasti v bujonu s 6,5 % NaCl, rezistence ob izpostavitvi temperaturi 60 °C za 15 in 30 minut, Voges-Proskauerjeve reakcije in fermentacije riboze. Izolirani sevi enterokokov so bili v 65,82 % primerov odporni proti penicilinu, 62,02 % sevov je bilo odpornih proti tetraciklinu, 68,35 % proti linkomicinu in 27,84 % proti gentamicinu. Odpornost proti neomicinu je bila prisotna pri 31,64 % proučevanih sevov, proti eritromicinu pri 31,64 % in proti kloramfenikolu pri 65,82 % sevov. Ključne besede: mlečni izdelki / sir / mikrobiologija / enterokoki / fenotipizacija / antibiotiki / rezistenca INTRODUCTION Enterococci are ubiquitous bacteria which colonize different niches. The primary habitat is considered to be the gastrointestinal tract of animals and humans, thus via fecal contamination reach the raw milk and meat. The wide distribution of the enterococci, their resistance to high temperatures, tolerance to high salt concentration argumented their survival through the production process and implication that this group of organisms, may also be isolated from heat-treated milk and dairy products, especially cheese made from raw milk. Enterococci, present in raw milk, may develop during cheese making process and ripening period and may represent the predominant microflora found in cheese made from raw milk (Neviani et al.,1982a, Neviani et al.,1982b). Depending on the stage of ripening, enterococci can reach numbers of up to 106–108 cfu g–1 (Fontecha et al., 1990; Basso et al., 1994). In cheeses like Manchego (Ordonez et al., 1978), Mozzarella (Coppola et al., 1988), Kefalotyri (Litopoulou-Tzanetaki, 1990), Feta and Teleme (Tzanetakis and Litopoulou-Tzanetaki, 1992), Serra (Macedo et al., 1995), Cebreiro (Centeno et al., 1996) and Comte (Bouton et al., 1998) enterococci represent a major part of the fresh cheese curd microflora and particularly they are the predominant microorganisms in the fully ripened cheese. In fresh Feta cheese, the normal microflora of lactic acid bacteria consists of starter cultures that are gradually replaced by salt resistant lactobacilli and enterococci, mainly Enterococcus faecalis and Enterococcus faecium (Tzanetakis et al., 1995).The predominant microorganism in Cebreiro cheese is Enterococcus faecalis, which is also the most frequently isolated Enterococcus in acidified raw milk (Wessels et al., 1988; Jiwoua and Milliere, 1990) and fresh Italian cheeses (Soncini and Piantoni, 1992). There are contradictory reports on the influence of enterococci on sensory characteristics of cheese. High contamination levels of enterococci are considered to cause the deterioration of organoleptic properties in some cheese (Thompson and Marth, 1986; Lopez-Diaz et al., 1995). On contrary, many authors claim that enterococci may have a positive role in cheese making process (Jensen et al., 1975b and 1975c; Ordonez et al., 1978; Trovatelli and Schliesser, 1987; Centeno et al., 1999). The high proteolytic activity presented by some strains of Enterococcus faecalis could contribute to the sensorial and textural properties of cheese (Centeno et al., 1999). In addition, enterococci produce esterases, which can play an important role in flavour formation (Tsakalidou et al., 1993). According to many authors, Enterococcus faecalis has been successfully used to accelerate maturation and to improve organoleptic characteristics of cheeses (Jensen et al., 1975a; Neviani et al. 1982b; Hegazi, 1990; Ledda et al., 1994;Villani and Coppola, 1994; Tzanetakis et al., 1995). On the basis of well-documented desirable biochemical properties, which argumented technological acceptability of enterococci, they have been proposed as part of defined starter cultures for different European cheeses, such as Water-Buffalo Mozzarella (Villani and Coppola, 1994), Feta (Litopoulou-Tzanetaki et al., 1993), Venaco (Casalta and Zennaro, 1997) and Cebreiro (Centeno et al., 1996) cheese. Bulajić, S. and Mijačević, Z. Enterococci in cheeses-phenotypization and antibiotic resistance. 27 Moreover, many enterococci produce one or more bacteriocins, and may be considered as protective towards spoilage and pathogenic bacteria (De Vuyst, 1994; Cintas et al., 1997; Aymerich et al., 2000; Giraffa, 1995). Furthermore, a strain of E. faecium SF68 has been confirmed as a probiotic according to its positive effects against diarrhea in man and pigs (Underdahl, 1983). But, in spite of all this, there is no consensus whether these bacteria pose the risk in food fermentation process, because of their ability to develop resistance against most antibiotics currently used, in combination with known virulence factors. The strains of enterococci are naturally tolerant to ß-lactams, cephalosporins, lincosamides and polymyxins. A specific cause for concern and a factor contributing to the pathogenesis of enterococci is the resistance they acquire to aminoglycosides, tetracyclines, macrolides, chloramphenicol, penicillin, and ampicillin (Gray et al., 1991), and their capacity to exchange genetic information by conjugation. This paper reports on enterococci, isolated from cheese originated from Serbia and its patterns of susceptibility to selected antibiotics. MATERIAL AND METHODS Cheese samples The material of investigation was consisted of samples of fresh and ripened cheeses made from raw and cooked milk subjected to rennet or acid coagulation. Microbiological analysis Ten grams of each sample were homogenized with sterile solution of sodium citrate (20 g l–1), adequately diluted in sterile Ringer solution and spread on kanamycin aesculine azide (KAA; Oxoid,) plates. After 24 h incubation at 42 °C in aerobic conditions, colonies that displayed the typical enterococcal growth and cell morphology were picked up and purified twice on KAA plates. The phenotypization of isolated strains was performed according to following tests: catalase activity, growth in MRS broth at 10 °C and 45 °C, growth at pH 9.6, growth in broth containing 6.5% NaCl, growth in 0.1% methylen blue milk, resistance at 60 °C/15 and 30 minutes, Voges/Proskauer reaction, and fermentation of ribose. Antibiotic resistance patterns Antibiotic resistance was tested by the agar diffusion method on plates of Muller-Hinton agar supplemented with antibiotic disks (BD BBL Sensi-Disc Antimicrobial Susceptibility Test Discs), according to the directions of the manufacturer of disks. RESULTS AND DISSCUSION The number of enterococci determined in cheese samples depended on applied technological process. In the samples of fresh, by rennet coagulated cheese made from raw and cooked milk, the number of enterococci was ranged from 8.0×104 to 9.0×106 cfu g–1, and from 4.0×107 to 4.4×107 cfu g–1, respectively. In the case of ripened cheese made from raw and cooked milk subjected to acid coagulation, the number of enterococci was ranged from <102 to 5.0×104 cfu g–1 and from 2.5×104 to 1.5×105 cfu g–1, respectively. In the samples of fresh cheese made from raw and cooked milk subjected to acid coagulation the number of enterococci was ranged from 2.4×105 to 2.12×107 cfu g–1, and from 5×104 to 6×104 cfu g–1, respectively. 28 Acta agriculturae slovenica, 84(december 2004)1. Similar enterococcal number determined in cheese samples was reported by Fontecha et al. (1990) and Basso et al. (1994). Teuber et al. (1999) highlighted the finding that the contaminating enterococci may multiply to high number, e.g. more than 1.107 cfu per gram in soft cheeses. A preliminary study of 67 European yielded 40% samples containing enterococci in the range from 103 to 107 cfu g–1 (Sievers et al. 1993). Table 1. Number of enterococci determined in cheese samples Preglednica 1. Število enterokokov v vzorcih sira N (cfu / g) Fresh cheeses Ripened cheeses Rennet coagulation Acid coagulation Acid coagulation Raw milk 8.0×104 to 9.0×106 2.4×105 to 2.12×107 <102 to 5.0×104 Cooked milk 4.0×107 to 4.4×107 5×104 to 6×104 2.5×104 to 1.5×105 Fourty two strains of enterococci were isolated, which were subjected to antibiotic susceptibility testing. The isolated strains of enterococci were resistant to following antibiotics: penicillin (65.82%), tetracycline (62.02%), lincomycin (68.35%), gentamycin (27.84%), neomycin (31.64%), erithromycin (31.64%), and chloramphenicol (65.82%). Sievers et al. (1993) reported that in 55% of analysed cheeses, E. faecalis, E. faecium, and E. durans strains were isolated showing resistance to one or more following antibiotics: penicillin, cefalotin, furadoin, fucidin, erytromycin, tetracycline and chloramphenicol. Antibiotic resistant enterococci were present in different food items, including raw milk cheese (Emmenthal, Appenzell, Gruyere, Tilsit and soft cheeses) (Baumargartner et al., 2001). In the same study, resistance to chloramphenicol, erytromycin and tetracycline was prominent in E. faecalis, while E. faecium showed resistance to ampicillin, nitrofurantoin, penicillin and tetracycline. In order to evaluate the potential risk which food contaminated with resistant strains of enterococci represent to human health, it is important to distinguish between intrinsic and acquired antibiotic resistance (Clewell, 1990; Murray, 1990). Further investigations are needed to address this question. REFERENCES Aymerich, T./ Artigas, M.G./ Garriga, M./ Monfort, J.M./ Hugas M. Effect of sausage ingredients and additives on the production of enterocins A and B by enterococcus faecium CTC492. Optimization of in vitro production and anti-listerial effect in dry fermented sausages. J. Appl. Microbiol., 88(2000), 686–694. Basso, A./ Goffo, A./ Rossi, G./ Conterno, N. A preliminary characterization of the microflora of Montasio cheese – Occurence of galactose fermenting strains in cheese and in natural starter cultures. Microbiologie - Aliments – Nutrition, 12(1994), 139–144. Baumgartner, A.// Kueffer, M./ Rohner, P. Occurence and antibiotic resistance of enterococci in various ready-to-eat foods. Arch Lebensmittelhyg., 52(2001), 1–24. Bouton, Y./ Guyot, P./ Grappin, P. Preliminary characterization of microflora of Comte cheese. Journal of Applied Microbiology, 85(1998), 123–131. Casalta, E./ Zennaro R. Effect of specific starters on microbiological, biochemical and sensory characteristics of Venaco, a Corsican soft cheese. Sci. Alim., 17(1997), 79–94. Centeno, J.A./ Menendez, S./ Hermida, M.A./ Rodriguez-Otero, J.L. Effects of the addition of Enterococcus faecalis in Cebreiro cheese manufacture. International Journal of Food Microbiology, 48(1999), 97–101. Centeno, J.A./ Menendez, S./ Rodriguez-Otero, J.L. Main microbial flora present as natural starters in Cebreiro raw cow’s milk cheese (Northwest Spain). International Journal of Food Microbiology, 33(1996), 307–313. Cintas, L.M./ Casaus, P./ Havarstein, L.S./ Hernandez, P.E./ Nes, I.F. Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum. Appl. Environ. Microbiol., 63(1997), 4321–4330. Clewell, D.B. Movable genetic elements and antibiotic resistance in enterococci. European Juornal of Clinical Microbiological Infection Diseases, 9(1990), 90–102. Bulajić, S. and Mijačević, Z. Enterococci in cheeses-phenotypization and antibiotic resistance. 29 Coppola, T.M./ Parente, J.E./ Dumontet, S./ La Peccerella, A. The microflora of natural whey cultures utilized as starters in the manufacture of Mozzarella cheese from water buffalo milk. Lait, 68(1988), 295–310. De Vuyst, L. Bacteriocins of Enterococcus. In: Bacteriocins of Lactic Acid Bacteria: Microbiology, Genetics and Applications (Eds.: De Vuyst, L./ Vandamme, E.J.). London, Blackie Academic and Professional, (1994), 511–513. Fontecha, J./ Pelaez, C./ Juares, M./ Requena, T./ Gomez, C. Biochemical and microbiological characteristics of artisanal hard goat’s cheese. Journal of Dairy Science, 73(1990), 1150–1157. Giraffa, G. Enterococcal bacteriocins:their potential as anti-Listeria factors in dairy technology. Food Microbiol., 12(1995), 291–299. Gray, J.W./ Stewart, D./ Pedler, S.J. Species identification and antibiotic susceptibility testing of enterococci isolated from hospitalized patients. Antimicrob. Agents Chemother., 35(1991), 1943–1945. Hegazi, F.Z. Extracellular proteinase of Enterococcus faecalis subsp. liquefaciens: production-milk-clotting and proteolytic activities. Microbiologie-Aliments-Nutrition, 8(1990), 341–348. Jensen, J.P./ Reinbold, G.W./ Washam, C.J./ Vedamuthu, E.R. Role of enterococci in Cheddar cheese: Organoleptic consideration J. Milk and Food Technology, 38(1975a), 142–145. Jensen, J.P./ Reinbold, G.W./ Washam, C.J./ Vedamuthu, E.R. Role of enterococci in Cheddar cheese: proteolytic activity and lactic acid development. Journal of Milk and Food Technology, 38(1975b), 3–7. Jensen, J.P./ Reinbold, G.W./ Washam, C.J./ Vedamuthu, E.R. Role of enterococci in Cheddar cheese: free fatty acid appearsnce and citric acid utilization. Journal of Milk and Food Technology, 38(1975c), 78–83. Jiwoua, C./ Milliere, J.B. Flore lactique et enterocoques du lait caill (Pindidam) produit dans l’Adamaoua (Cameroun). Lait, 70(1990), 475–486. Ledda, A./ Scintu, M.F./ Pirisi, A./ Sanna, S./ Mannu, L. Caratterizzazione tecnologica di ceppi di lattococchi e di enterococchi per la produzione di formaggio pecorino Fiore sardo. Scienza e Tecnica Lattiero Casearia, 45(1994), 443–456. Litopoulou-Tzanetaki, E. Changes in numbers and kinds of lactic acid bacteria during ripening of Kefalotyri cheese. Journal of Food Science, 55(1990), 111–113. Litopoulou-Tzanetaki, E./ Tzanetakis N./ Vafopoulou-Mastrojiannaki A. Effect of type of lactic starter on microbiological, chemical and sensory characteristics of Feta cheese. Food Microb., 10(1993), 31–34. Lopez-Diaz, T.M./ Santos, J.A./ Gonzales, C.J./ Moreno, B./ Garcia, M.L. Bacteriological quality of traditional Spanish blue cheese. Milchwissenschaft, 50(995), 503–505. Macedo, C.A./ Malcata, F.X./ Hogg. T.A. Microbiological profile in Serra ewe’s cheese during ripening. Journal of Applied Bacteriology, 79(1995), 1–11. Murray, B.E. The life and times of enterococcus. Clinical Microbiological Reviews, 3(1990), 46–65. Neviani, E./ Mucchetti, G./ Contarini, G./ Carini, S. Ruolo delle Enterococcaceae nei formaggi italiani. II Latte, 7(1982b), 722–728. Neviani, E./ Mucchetti, G./ Contarini, G./ Garini, S. Ruolo degli enterococci nei formaggi italiani. I. Loro presenza in formaggi di monte e impiego in un innesto selezionato. Latt, 7(1982a), 722–728. Ordonez, J.A./ Barneto, R/ Ramos, M. Studies on Manchego cheese ripened in olive oil. Milchwissenschaft, 33(1987), 609–612. Sievers, M./ Teuber, M./ Wirsching, F./ Perreten, V./ Eisenring, R./ Fahndrich, P./ Schlaepfer, S./ Krusch, U./ Simm, Ch./ Lohmer, A. Antibiotic resistance properties of enterococci and staphyloicocci from fermented food. Food Micro '93. Book of Abstracts, 191 p. Soncini, G./ Piantoni, L. Enterococchi: isolamento e identificazione di ceppi evidenziati in formaggi di monte. II Latte, 17(1992), 1139–1141. Teuber, M./ Meile, L./ Schwarz F. Acquired antibiotic resistance in lactic acid bacteria from food. Antonie van Leeuwenhoek, 76(1999), 115–137. Thompons, T.L./ Marth, E.H. Changes in Parmesan cheese during ripening: Microflora - coliforms, enterococci, anaerobs, propionibacteria and staphylococci. Milchwissenschaft, 41(1986), 201–204. Trovatelli, L.D./ Schliesser, A. Identification and significance of enterococci in hard cheese made from raw cow and sheep milk. Milchwissenschaft, 42(1987), 717–719. Tsakalidou, E./ Manolopoulou, E./ Tsilibari, V./ Georgalaki, M./ Kalantzopoulos, G. Esterolytic activities of Enterococcus durans and E. faecium strains isolated from Greek cheese. Netherlands Milk and Dairy Journal, 47(1993), 145–150. Tzanetakis, N./ Litopoulou-Tzanetaki, E. Changes in numbers and kind of lactic acid bacteria in Feta and Teleme, two Greek cheeses from ewe’s milk. Journal of Dairy Science, 75(1992), 1389–1393. Tzanetakis, N./ Vafopoulou-Mastrojiannaki, A./ Litopoulou-Tzanetaki, E. The quality of white-brined cheese from goat’s milk made from different starters. Food Microbiol., 12(1995), 55–63. Underdahl, N.R. The effect of feeding Streptococcus faecium upon Escherichia coli induced diarrhoea in gnotobiotic pigs. Progress in Food and Nutritional Science, 7(1983), 5–12. 30 Acta agriculturae slovenica, 84(december 2004)1. Villani, F./ Coppola, S. Selection of enterococcal strains for water-buffalo Mozzarella cheese manufacture. Annali di Microbiologia ed Enzimologia, 44(1994), 97–105. Wessels, D./ Jooste, P.J./ Mostert, J.F. Die voorkoms van Enterococcus-spesies in melk end suiwelprodukte. Suid-Afrikaanse Tydskrif vir Suiwelkunde, 20(1988), 68–72. Acta agriculturae slovenica, 84(december 2004)1, 31-36. http://www.bfro.uni-lj.si/zoo/publikacije/zbornik Agris category codes: Q01 COBISS Code 1.01 ENUMERATION, ISOLATION, AND IDENTIFICATION OF BIFIDOBACTERIA FROM DAIRY PRODUCTS Eva VLKOVÁ a), Vojtěch RADA b) and Iva TROJANOVÁ a) a) Department of Microbiology and Biotechnology, Czech University of Agriculture Prague, Kamýcká 129, 165 21 Prague 6, Czech Republic. b) Same address, Ass. Prof. Received June 10, 2004, accepted October 15, 2004. Delo je prispelo 10. junija 2004, sprejeto 15. oktobra 2004. ABSTRACT Six dairy products were tested. Bifidobacteria were enumerated and isolated using TPY agar modified by the addition of mupirocin (100 mg l–1). Isolates were identified to the genus level by the detection of fructose-6-phosphate phosphoketolase (F6PPK) and by the FISH method. Bifidobacteria were characterised using API 50 CHL and API ID 32 A Rapid tests. In addition, grow at 46 °C was tested. Subsequently, all strains were identified to the species level using computer program Bacter. The identification was also carried out by PCR method using genus-and species-specific primers. The bifidobacterial counts in products tested varied from 2.37 to 7.17 log CFU/ml. High selectivity was seen for modified TPY agar from which all isolates were identified as bifidobacteria. While most strains were identified as B. animalis using Bacter program, the same isolates had positive reaction with B. lactis-specific primers. Strain isolated from Rajo yogurt was identified as B. longum by Bacter as well as using PCR, but its bacterial counts were too low. Our results showed, that most of the bifidobacterial strains currently used in food products could be of animal origin. Key words: milk products / dairy products / microbiology / bifidobacteria / enumeration / identification / isolation ŠTETJE, IZOLACIJA IN IDENTIFIKACIJA BIFIDOBAKTERIJ V MLEČNIH IZDELKIH IZVLEČEK Testirali smo šest mlečnih izdelkov. Štetje bifidobakterij in izolacijo smo izvedli na TPY agarju, ki smo mu dodali muriprocin (100 mg l–1). Izolate smo identificirali na ravni rodu z detekcijo fruktoza-6-fosfat fosfoketolaze (F6PPK) in z metodo FISH. Bifidobakterije smo opisali s hitrima testoma API 50 CHL in API ID 32. Preverili smo tudi rast pri 46 °C. Nato smo vse isolate identificirali na ravni vrst z računalniškim programom Bacter. Dodatno smo izvedli identifikacijo z metodo PCR ob uporabi za posamezen rod, oziroma vrsto specifičnih začetnih oligonukleotidov. Število bifidobakterij v izdelkih je variralo med 2,37 in 7,17 log ke/ml. Opazili smo visoko selektivnost modificiranega TPY agarja, na katerem smo izolirali izključno bifidobakterije. Kljub temu, da smo večino izolatov z računalniškim programom Bacter identificirali kot B. animalis, smo DNA nekaterih izolatov lahko pomnožili z začetnimi oligonukleotidi, specifičnimi za B. lactis. Izolat iz jogurta Rajo smo s programom Bacter in vrstno specifično PCR identificirali kot B. longum, vendar je bilo število celic zelo nizko. Naši rezultati kažejo, da bi bila lahko večina sevov bifidobakterij, ki so v uporabi prehranski industriji, živalskega izvora. Ključne besede: mlečni izdelki / mikrobiologija / bifidobakterije / štetje / identifikacija / izolacija 32 Acta agriculturae slovenica, 84(december 2004)1. INTRODUCTION Bifidobacteria are Gram-positive, non-sporeforming, non-motile, anaerobic, irregular rods. The typical habitat of bifidobacteria is human, warm-blooded animal and honeybee intestinal tract (Scardovi, 1986). Members of genus Bifidobacterium (B.) are among the most common microorganisms in the human gut, comprising up to 3% of the total faecal microflora of adults (Sghir et al., 2000). They are more numerous in the infant gut, where they form up to 91% of the total microflora in breast-fed babies being supported by bifidogenic factors presented in human milk and up to 75% in formula-fed infants (Harmsen et al., 2000). Using classical culturing methods it has been found that B. adolescentis and B. longum are major bifidobacterial species in the adult intestine (Gavini et al., 2001; Biavati et al., 1986; Mutai and Tanaka, 1987) and that B. infantis and B. breve are predominant species in the intestinal tract of human infants (Benno et al., 1984; Biavati et al., 1984; Mutai and Tanaka, 1987). In addition, B. bifidum, B. catenulatum, B. pseudocatenulatum, B. angulatum, B. gallicum, and B. dentium have also been reported to be human intestinal bifidobacteria (Scardovi, 1986). Matsuki et al. (1999) who used for the detection of bifidobacteria in human gut species-specific polymerase chain reaction (PCR) reported, that the most common species in the breast-fed infants are B. breve, B. infantis, B. longum, and B. bifidum. In adult intestinal tracts, the B. catenulatum group was the most common taxon, followed by B. longum and B. adolescentis. The genus Bifidobacterium constitutes a significant proportion of the probiotic cultures used in the food industry (Langhendries et al., 1995; Saavedra et al., 1994). The employment of strains belonging to B. animalis, B. longum, B. bifidum, and B. infantis as probiotic starter cultures is due to their important role played in the gut (Gibson and Roberfroid, 1995; Modler et al., 1990). They suppress harmful bacteria by controlling pH of the large intestine through the production of lactic and acetic acids (Gibson et al., 1997). Bifidobacteria have antitumoral activity (Reddy and Rivenson, 1993; Rastall and Gibson, 2002), anticholesterolemic (Pereira and Gibson, 2002), and immune system activation effects (Mitsuoka, 1992). Other effects that have been described to this genus are the alleviation of lactose intolerance and vitamin production (Hughes and Hoover, 1995; Fooks et al., 1999). The presence of high number of bifidobacteria in the large intestine is desirable and can be influenced by dietary supplementation with probiotics and/or prebiotics. Probiotics have been defined as living microorganisms, which beneficially affect the host by improving its intestinal microbial balance (Fuller, 1989). Prebiotics are nondigestible dietary supplements that modify the balance of the intestinal microflora, stimulating the growth and/or activity of beneficial organisms and suppressing potentially deleterious bacteria (Gibson and Roberfroid, 1995). In order to exert a beneficial effect, probiotic bacteria should be viable and present at high numbers in the product at time of consumption (McBrearty et al., 2001). Industrial interest in the use of bifidobacterial strains as food additives in dairy products is rapidly growing. This development leads to the requirement for accurate quantity and quality control of the probiotic products and hence methods for specific identification of probiotic strains. Consequently, the aim of our work was to enumerate and isolate bifidobacteria from dairy products, and to compare biochemical and molecular biology methods for the identification of these isolates. MATERIAL AND METHODS Six dairy products were tested, three yogurts and three fermented milk products. Five products were made in the Czech Republic and one (Rajo) in the Slovak republic. The list of products tested is in Table 1. Vlková, E. et al. Enumeration, isolation, and identification of bifidobacteria from dairy products. 33 Table 1. Tested products Product Product type Made in Activia yogurt Czech Republic Hollandia yogurt Czech Republic Olma – Dr. Bio fermented milk product Czech Republic Yoplait fermented milk product Czech Republic Kefir-like milk fermented milk product Czech Republic Rajo yogurt Slovak Republic Bifidobacteria in dairy products were enumerated and isolated using TPY agar (Sharlou, Barcelona, Spain) modified by the addition of mupirocin at a concentration of 100 mg/L (Rada and Koc, 2000). Pure isolates were enriched in TPY broth, and were identified as members of the genus Bifidobacterium by the demonstration of fructose-6-phosphate phosphoketolase (EC 4.1.2.22) activity, as described by Orban and Patterson (2000). The genus identification was performed also using fluorescence in situ hybridisation (FISH) kit for Bifidobacterium sp. (RiboTechnologies, Groningen, the Netherlands). The component of the kit is a genus-specific oligonucleotide DNA probe labelled by fluorescein isothiocyanate (FITC), which binds to bifidobacterial rRNA. After the hybridization, the samples were analysed with a Nikon E-800 epifluorescence microscope. Another method used for the genus identification was PCR with genus specific primers which was performed as described previously (Kok et al., 1996). All isolates were tested for the ability to grow at 46 °C. Testing of grow at this temperature is a method recommended for distinguishing of human and animal strains. Human isolates are not able to grow at 46 °C and most of animal isolates are able to grow at this temperature (Gavini et al., 1991). Subsequently, the isolates were characterised using API 50 CHL and API ID 32 A Rapid kits (BioMérieux, France). On the basis of the results from these tests, all strains were identified to the species level using computer program Bacter (http://kounou.lille.inra.fr, INRA, Lille, France). The identification to the species level was also carried out by PCR method using species-specific primers. The genomic DNA of the strains was extracted by heating at 100 °C for 10 minutes in 1% Triton X-100 (Sigma, USA) by the method described by Wang et al. (1996). Amplifications were performed with a thermal cycler (Techne, Techgene, UK) with solutions, species-specific primers and temperature profiles described by Matsuki et al. (1999). Amplified PCR products were analyzed by 1% agarose gel electrophoresis at a constant voltage of 7 V.cm–1 and visualized with ethidium bromide (0.5 µg/mL) under UV light (wavelength, 260 nm). RESULTS AND DISCUSSION Bifidobacterial counts determined in tested products are shown in Table 2. The counts varied from 2.37 to 7.17 log CFU/mL. Recommended lower limit of International Dairy Federation (IDF) for bifidobacterial counts in dairy product is 106 CFU per one mL. In Japan this recommendation is even at least 107 viable probiotic cells per gram or millilitre (Ishibashi and Shimamura, 1993). Generally, bifidobacteria show poor viability in fermented dairy products and various studies have indicated that not all probiotic products contain the recommended levels of viable microorganisms (Kailasapathy and Rybka, 1997; Dave and Shah, 1997). Only four of our product tested fulfil the recommendation of IDF, while two products did not meet these criteria. In Rajo yogurt the counts of bifidobacteria were only 2.37 log CFU/mL. 34 Acta agriculturae slovenica, 84(december 2004)1. High selectivity was observed for modified TPY agar from which all isolates were F6PPK-positive and were identified as bifidobacteria. These results were confirmed by FISH and PCR methods. Table 2. Bifidobacterial counts and species isolated from dairy products Product Bifidobacterial counts Species isolated Activia 7.17 ± 0.09a B. animalis/B. lactis Hollandia 6.31 ± 0.38a B. animalis/B. lactis Olma – Dr. Bio 6.22 ± 0.20a B. animalis/B. lactis Yoplait 6.03 ± 0.12a B. animalis/B. lactis Kefir-like milk 5.44 ± 0.41b B. animalis/B. lactis Rajo 2.37 ± 0.47c B. longum Results are means (n=3) ± SD of log CFU/mL a,b,cData in column with no common superscripts differ (P < 0.05) While five strains were identified as B. animalis using Bacter program, the same isolates had positive reaction with B. lactis-specific primers. Hence, it is not clear when B. lactis and B. animalis are the same species, because Bacter database does not contain species B. lactis and on contrary, there are no available B. animalis-specific primers. Cai et al. (2000) reported that the relative taxonomic position of B. lactis is still under discussion and that B. lactis could be considered a junior subjective synonym for B. animalis. In contrary, Ventura et al. (2001) demonstrated clear differences in rDNA sequences between B. lactis (DSM 10141) and the type strain of B. animalis (ATCC 25227). A decision of this issue by the International Committee on Systematic Bacteriology is still outstanding (Anonym, 2001). Strain isolated from Rajo yogurt (1?) was identified as B. longum, which is the human origin species, by both Bacter as well as using PCR. But its bifidobacterial counts were too low. Five from six dairy-related isolates were identified as species of animal origin, although it is recommended that the bifidobacterial strains used in fermented milk products should be of human origin. Especially B. animalis is often found in dairy products (Bonaparte, 1997). A modified TPY agar was found to be highly selective and suitable for isolation and enumeration of bifidobacteria from dairy products, as all isolates in our study were identified as bifidobacteria. Our results also showed that most of the bifidobacterial strains currently used in food products are probably of animal origin. Only one strain was identified as B. longum, which is of human origin, but its survival in fermented milk product was poor. Further investigations should be focused on the selection of human bifidobacterial isolates which are able to survive in milk for longer period of time. Also, the identity and origin of currently used strains should be clearified. This study was supported by grants numbers MSM 412100003, 1454/G4, and 1425/G4 of the Grant Agency of Ministry of Education, Youth and Sports of Czech Republic, and 523/03/H076 of the Grant Agency of Czech Republic. REFERENCES: Anonym. International Committee on Systematic Bacteriology. Veldhoven, The Netherlands. Int. J. Syst. Evol. Microbiol., 51(2001), 259–261. Benno, Y./ Sawada, K./ Mitsuoka, T. The intestinal microflora of infants: composition of fecal flora in breast-fed and bottle-fed infants. Microbiol. Immunol., 28(1984), 975–986. Biavati, B./ Catagnoli, P./ Crociani, F./ Trovatelli, L.D. Species of the Bifidobacterium in the feces of infants. Microbiologica., 7(1984), 341–345. Vlková, E. et al. Enumeration, isolation, and identification of bifidobacteria from dairy products. 35 Biavati, B./ Catagnoli, P./ Trovatelli, L.D. Species of the Bifidobacterium in the feces of human adults. Microbiologica., 9(1986), 39–45. Bonaparte, S. Selective isolation and taxonomic position of bifidobacteria isolated from commercial fermented dairy products in central Europe. PhD Thesis, Münster, Berlin Technical University, 1997, 199 p. Cai, Y./ Matsumoto, M./ Benno, Y. Bifidobacterium lactis Meile et al. 1997 is a subjective synonym of Bifidobacterium animalis (Mitsuoka 1969) Scardovi and Trovatelli 1974. Microbiol. Immunol., 44(2000), 815–820. Dave, R.I./ Shah, N.P. Viability of yoghurt and probiotic bacteria in yoghurts made from commercial starter cultures. Int. Dairy J., 7(1997), 31–41. Fooks, L.J./ Fuller, R./ Gibson, G.R. Prebiotics, probiotics and human gut microbiology. Int. Dairy. J., 9(1999), 53–61. Fuller, R. Probiotic in man and animal. J. Appl. Bacteriol., 66(1989), 35–378. Gavini, F./ Cayuela, Ch./ Antoine, J.M./ Lecoq, C./ Lefebvre, B./ Membré, J.M./ Neut, Ch. Differences in the distribution of bifidobacterial and enterobacterial species in human faecal microflora of three different (children, adults, elderly) age groups. Microb. Ecol. Health. Dis., 13(2001), 40–45. Gavini, F./ Pourcher, A.M./ Neut, C./ Monget, D./ Romond, C./ Oger, C./ Izard, D. Phenotypic differentiation of bifidobacteria of human and animal origin. Internat. J. Syst. Bacteriol., 41(1991), 548–557. Gibson, G.R./ Roberfroid, M.B. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J. Nutr., 125(1995), 1410–1412. Gibson, G.R./ Saaverda, J.M./ MacFarlane, S./ MacFarlane, G.T. Probiotics and intestinal infections. In: Probiotics 2: Application and Practical Aspects (Ed.: Fuller, R.). London, Chapman and Hall, 1997, 10–31. Harmsen, H.J.M./ Wildehoer, A.C.M./ Raangs, G.C./ Wagendorp, A.A./ Klijn, N./ Bindels, J.G./ Welling, G.W. Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J. Pediatr. Gastroenterol. Nutr., 30(2000), 61–67. Hughes, B.B./ Hoover, D.G. Viability and enzymatic activity of bifidobacteria in milk. J. Dairy. Sci., 78(1995), 268–276. Ishibahi, N./ Shimamura, S. Bifidobacteria: Research and development in Japan. Food. Technol., 6(1993), 126–135. Kailasapathy, K./ Rybka, S. L. acidophilus and Bifidobacterium spp. - their therapeutic potential and survival in yogurt. Austr. J. Dairy Technol., 52(1997), 28–35. Kok, R.G./ De Waal, A./ Schut, F./ Welling, G.W./ Week, G./ Hellingwerf, K.J. Specific detection and analysis of a probiotic Bifidobacterium strain in infant feces. Appl. Environ. Microbiol., 62(1996), 3668–3672. Langhendries, J.P./ Detry, J./ van Hees, J./ Lamboray, J.M. Effect of a fermented infant formula containing viable bifidobacteria on the fecal flora composition and pH of healthy full-term infants. J. Pediatr. Gastroenterol. Nutr., 21(1995), 177–181. Matsuki, T./ Watanabe, K./ Tanaka, R./ Fukuda, M./ Oyaizu, H. Distribution of bifidobacterial species in human intestinal microflora examined with 16S rRNA-gene-targeted species-specific primers. Appl. Environ. Microbiol., 65(1999), 4506–4512. McBrearty, S./ Ross, R.P./ Fitzgerald, G.F./ Collins, J.K./ Wallace, J.M./ Stanton, C. Influence of two commercially available bifidobacteria cultures on Cheddar cheese quality. Int. Dairy J., 11(2001), 599–610. Mitsuoka, T. The Human Gastrointestinal Tract. In: The Lactic Acid Bacteria in Health and Disease (Ed.: Wood, B.J.B.). London, Elsevier Applied Science, 1992, 69–114. Modler, H.W./ McKellar, R.C./ Yaguchi, M. Bifidobacteria and bifidogenic factors. Can. Inst. Food. Sci. Technol. J., 23(1990), 29–41. Mutai, M./ Tanaka, R. Ecology of Bifidobacterium in the human intestinal flora. Bifidobacteria Microflora, 6(1987), 33–61. Pereira, D.I.A./ Gibson, G.R. Cholesterol assimilation by lactic acid bacteria and bifidobacteria isolated from the human gut. Appl. Environ. Microbiol., 98(2002), 4689–4693. Orban, J.I./ Patterson, J.A. Modification of the phosphoketolase assay for rapid identification of bifidobacteria. J. Microbiol. Meth., 40(2000), 221–224. Rada, V./ Koc, J. The use of mupirocin for selective enumeration of bifidobacteria in fermented milk products. Milchwissenschaft., 55(2000), 65–67. Rastall, R.A./ Gibson, G.R. Prebiotic Oligosaccharides: Evaluation of Biological Activities and Potential Future Developments. In: Probiotics and Prebiotics (Ed.: Tannock, G.W.). Norfolk, Caister Academic Press, 2002, 107–148. Reddy, B.S./ Rivenson, A. Inhibitory effect of Bifidobacterium longum on collon, mammary and liver carcinogenesis induced by 2-amino-3-methylimidaz (4, 5-f) quinoline, a food mutagen. Cancer Res., 53(1993), 3914–3918. Saavedra, J.M./ Abi-Hanna, A./ Moore, N./ Yolken, R. Effect of long term consumption of infant formulas with bifidobacteria and S. thermophilus on stool pattern and diaper rash in infants. J. Pediat. Gastroenerol. Nutr., 27(1998), 483–486. 36 Acta agriculturae slovenica, 84(december 2004)1. Scardovi, V. Genus Bifidobacterium. In: Bergey‘s Manual of Systematic Bacteriology, Vol. 2 (Eds.: Sneath, P.H.A./ Mair, N.S./ Sharpe, M.E./ Holt, J.G.). Baltimore, Williams and Wilkins, 1986, 1418–1434. Sghir, A./ Gramet, G./ Suau, A./ Rochet, V./ Pochart, P./ Dore, J. Quantification of bacterial groups within the human fecal flora by oligonucleotide probe hybridization. Appl. Environ. Microbiol., 66(2000), 2263–2266. Ventura, M./ Reniero, R./ Zink R. Specific identification and targeted characterization of Bifidobacterium lactis from different environmental isolates by a combined multiplex-PCR approach. Appl. Environ. Microbiol., 67(2001), 2760–2765. Wang, R./ Cao, W./ Cerniglia, C.E. PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples. Appl. Environ. Microbiol., 62(1996), 1242–1247. Acta agriculturae slovenica, 84(december 2004)1, 37-42. http://www.bfro.uni-lj.si/zoo/publikacije/zbornik Agris category codes: Q03 COBISS Code 1.01 DIAGNOSTIC USE OF PROFICIENCY TESTING IN DAIRY LABORATORY Oto HANUŠ a), Václava GENČUROVÁ b), Pavel HERING c), Miloš KLIMEŠ c) and Radoslava JEDELSKÁ b) a) Research Institute for Cattle Breeding, Ltd., Rapotin, Vyzkumniku 267, 788 13 Vikyrovice, Czech Republic, Ph.D. b) Same address as a). c) Czech-Moravia Breeders Association, a.s., Prague, 252 09 Hradistko pod Mednikem, Czech Republic. Received June 03, 2004, accepted October 15, 2004. Delo je prispelo 03. junija 2004, sprejeto 15. oktobra 2004. ABSTRACT The composition of raw milk (RM) is important for milk recording, herd improvement, payment of milk and for quality evaluation. The reliability of routine analytical results is therefore significant. A mistake occurrence could jeopardize the efficiency of results. Milk laboratories (ML) perform the systems of analytical quality assurance. Proficiency tests are carried out usually by 10 RM samples with modified variability of components. At evaluation the system of Euclidian distance (Re=( d 2+sd2)0,5) for participants order according to their analytical result reliability is used. Two specificaly modified types (designs) of systematic diagnostical graphs were constructed on the Re basis. The first type of flow diagnostical diagram for RM analysators is based on Shewhart´s diagram principles for Re parameter (alternating system of result rendering before and after calibrations). The second type is based on analysator set comparisons before (proficiency test results) and after calibration (success of calibration) by Re. The opposite comparison is possible and valuable as well. It makes possible the diagnosis and incidental coordinating of the corrections in the case of the unconformity occurrence. The positive contributions of the developed diagnostic graphics system are expected to: – improvement of estimation of unconformity sources of RM routine analytical results, their origin and character; – determination and improvement of incidental result corrections and instrument repairs; – improvement of reliability of routine results of RM analyses in general. Key words: milk / dairy laboratories / proficiency testing / quality DIAGNOSTIČNA UPORABA PREVERJANJA USPOSOBLJENOSTI MLEKARSKIH LABORATORIJEV IZVLEČEK Sestava surovega mleka (SM) je pomembna za kontrolo proizvodnje, selekcijo, plačevanje in vrednotenje kakovosti mleka. Zanesljivost rutinskih metod je ključnega pomena, ker bi napake ogrozile verodostojnost rezultatov. Mlekarski laboratoriji vpeljujejo sisteme za zagotavljanje kakovosti analitskih postopkov. Preverjanje usposobljenosti se izvaja navadno na 10 vzorcih SM z različno vsebnostjo osnovnih sestavin. Za preverjanje zanesljivosti analitskih rezultatov se pogosto uporablja sistem Evklidskih distanc (Re=( d 2+sd2)0,5). Na osnovi Re sta bili zasnovani dve modifikaciji modela diagnostičnih grafov. Prvi tip diagramov poteka za analizo SM temelji na Shewhart ovem diagramu za parameter Re (sistem za določitev rezultata pred in po kalibraciji). Drugi tip temelji na primerjavi serij rezultatov pred in po kalibraciji z uporabao Re. Primerjava omogoča diagnozo in korekcije v primeru neskladja rezultatov. Od razvoja diagnostičnega grafičnega sistema si obetamo izboljšano določitev virov in narave neskladnosti analitskih rezultatov rutinske kontrole sestave SM, izboljšanje in korekcij rezultatov in instrumentov ter izboljšanje zanesljivosti rutinski rezultatov analize SM na splošno. Ključne besede: mlekarstvo / laboratoriji / usposobljenost / preverjanje / mleko / kakovost 38 Acta agriculturae slovenica, 84(december 2004)1. INTRODUCTION The main composition and properties of raw milk and their testing systems are very important: – for performance of the milk recording; – for monitoring of dairy cow health state and controlling (prevention and treatments of the dairy cow production disorders); – for milk quality evaluation; – for animal genetic appreciation and improvement (cattle breeding); – for milk quality payment; – for dairy processing; – for foodstuff chain safety in general. The milk foodstuff chain is one of the most monitored and controlled foodstuff chains in terms of: – the number of routinely checked milk parameters (microbiological, compositional, technological); – regularity and relatively high frequency of the mentioned investigations; – mostly biological character of the mentioned investigations. In accordance with such information, there are assumptions, that the milk foodstuff chain could be one of the most safe of all known foodstuff chains. Therefore, the reliability of the referential and routine milk analytical results is very important as well. Incidental mistake occurrence could jeopardize the efficiency of the dairy production. In the framework of supporting of the mentioned facts the milk laboratories carry out the accreditation according to the international standard (EN ISO/IEC 17025) in the Czech Republic. The referential and routine milk laboratories performs the systems of the analytical quality assurance of the calibrations and measure functions of the master and routine milk analysators as well. It means, laboratories improve the analytical results reliability runningly. A similar situation is at using of all referential and routine milk analytical methods in general. The last development works about above mentioned topics are introduced in many scientific and professional papers (Grappin, 1985, 1993; Hanuš and Ficnar, 1990; Hanuš and Kaššovicová, 1992; Leray, 1993; Wood, 1994; Golc-Teger, 1996, 1997; Hanuš et al., 1996, 1998, 2000, 2001, 2002; Klopčič et al., 1999). Mentioned systems are currently developed and improved also in the framework of the projects MZe-ČR, NAZV, QF 3019 and MŠMT-ČR, INGO, LA103. MATERIAL AND METHODS The Czech milk referential and routine laboratories (laboratories of the milk recording = individual milk samples; central laboratories = bulk milk samples) take part regularly in the interlaboratory proficiency testing on the national and international levels as well. RICB Rapotin organizes proficiency testing of the routine milk recording laboratories for some raw milk components: fat (F); protein (P); lactose (L); urea (U). Tests are performed on the basis of ten raw milk samples with necessary modified variability of the relevant introduced components. The calculation system of the Euclidian distance to the origin (Re; according to Leray, 1993; Fig. 1) is used. Euclidian distance represents the distance of each laboratory to the origin (0,0) corresponding to the reference. Re is used for evaluation of the proficiency testing results and for order of the participants (laboratories) according to accuracy and reliability of their analytical results for the mentioned purposes. The Euclidian distance (Leray, 1993) from the origin calculation system is prefered for the mentioned purposes in comparison for instance to the often used Z-score system (Wood, 1994) because of its advantage to separate an incidental analytical mistake into two parts: – systematic error part and random error part. Such a differentiation ability could be important for suggested necessary diagnostical purposes at analytical mistake investigation, identification and specification. The design of diagnostical diagram system for a better identification of mistakes in milk analysis and specification was proposed, created and developed according to: – results of twenty two proficiency tests performed during the year (2002 and 2003) by RICB referential milk laboratory; – knowledge of result variability of reliability parameter (Re) in regularly (monthly) performed proficiency tests; – knowledge of result dynamics of reliability parameter in regularly Hanuš, O. et al. Diagnostic use of proficiency testing in dairy laboratory. 39 performed proficiency tests; – knowledege and consideration about interpretation efficiency of different evalution systems (such as Re, Z-score, Youden plot, Shewhart´s diagram) for interlaboratory proficiency testing results in terms of their real graphical ability to analytical mistake investigation, identification and specification. Figure 1. The expression system of Euclidian distance to the origin (Re) as accuracy parameter of milk analytical results in the proficiency testing (according to Leray, 1993). RESULTS AND DISCUSSION The diagnostical diagram systems for raw milk analysators were constructed as specific purpose modifications on the basis of the Shewhart´s diagram principles (Kupka, 1997) for the mentioned Re system (Fig. 2) and Re time diagram reciprocal comparisons (Fig. 3). The Re discrimination limits (HDLRe) were calculated for F, P, L and U according to long-term analytical result rows (2 years = 22 tests at month intervals) for Fig. 2 and according to results of instrument sets of proficiency tests and calibration process (Fig. 3; the half circle lines). The graphs of the individual instruments (Fig. 2), and calibration process and proficiency test comparisons (Fig. 3) are accessible for workers of the routine milk laboratories (workers of laboratory network) in the electronical way: - the Re situations (Fig. 2) before and after calibration are shown by the alternating system of the symbols (!×!×…). It makes possible the diagnose and incidental co-ordinating of the necessary corrections in the case of the result unconformity occurrence. It is possible to show separately two components of the instrument result reliability (Re): the mean difference ( d ); the variability of the differences (sd); by the same type of the graph as well. Such projection improves the diagnosis of incidental occurrence of the result error in terms of the effect estimation of the systematical and random error parts on the total 40 Acta agriculturae slovenica, 84(december 2004)1. value of the unconformity. There are the protein graphs chosen for four types of the instruments (Fig. 2) for instance: master instrument of the referential-routine laboratory system (network) with good stability in the referential laboratory; routine instrument with good stability in the routine laboratory; routine instrument with usual stability in the routine laboratory; routine instrument with unconformity occurrence; further, the mentioned modificated system shows the situations of the whole set of the analysators according to Re (Re=( d 2+sd2)0,5; by the original Re rendering (according to Leray, 1993) Fig. 1) by the mutual comparison of two graphs after the last calibration and before the next calibration (×!). The opposite comparison (!×) is possible and very valuable for relevant analytical mistake diagnoses as well (Fig. 3). Such rendering improves the further diagnostical possibilities for investigation, identification and specification of incidental unconformities. A Re 0.120 0.100 -0.080 -0.060 -0.040 0.020 0.000 10 15 20 25 30 35 number of test B Re 0.120 - 0.100 - 0.080 - 0.060 0.040 0.020 0.000 0 5 10 15 20 25 30 35 number of test C Re 0.120 -0.100 -0.080 -0.060 0.040 0.020 -0.000 - 0 5 10 15 20 25 30 35 number of test D Re 0.300 -0.250 -0.200 -0.150 0.100 0.050 -0.000 - X O ° y X x °x x x x0 oxox x x x 0 5 10 15 20 25 30 35 number of test Re = Euclidian distance from the origin (parameter of the result accuracy or reliability); HDLRe = historical discrimination limit of Re (robust median-quartile estimation of confidence interval at level 95%); × = situation after calibration (verifycation of the previous calibration results made immediately after calibration); O = situation before calibration (result of the ring proficiency test); Figure 2. Individual diagnostical flow graphs for protein content at instruments (A,B,C,D) with different measurement stability. The results of the proficiency testing are anonymous, of course. Nevertheless, if the laboratory managers know the key numbers of their own instruments and further they can compare the result reliability (accuracy) situations by different types of mentioned developped diagnostical graph combinations, they could be able to guess the type, character and source of 0 5 HDLRe Hanuš, O. et al. Diagnostic use of proficiency testing in dairy laboratory. 41 incidental analytical mistakes according to their own knowledge about analytical method principles and simultaneous laboratory facts. CONCLUSIONS It is expected, that the mentioned developped diagnostical graphical system for the analytical mistake identification and speification will contribute positively in the milk referential-routine laboratory networks to: improve of the estimation of unconformity sources of the raw milk routine analytical results in terms of their origin and character; determine and improve the incidental corrections of the results and necessary reparations of the instruments; improve the quality (reliability) of the routine results of the raw milk analyses in general. Fat After calibration, success of performed calibration One month later, before next calibration, result of proficiency testing sd 0.060- 0.050- _^0.040 ~~~^ * 0.030* X Xx x\ X / 0020" 0.010- X / X \ -0.060 -0.040 -0.020 0.000 0.020 0.040 0.060 0.080 -, 0.060 - o x^y , 0.050 - \ 0 0 \ 0.040 - 0.030 - IWWWW\ acid producers, kislinotvorni m.o. 024 no. of microorganisms, in log št. Mikroorganizmov, v log 10 Figure 5. The average number (in log) of different groups of micro-organisms expressed as the number of colony-forming units cfu per 1 ml of brine samples. Slika 5. Povprečno število (v log) mikroorganizmov različnih skupin, izraženo kot število kolonijskih enot ke v 1 ml vzorca slanice. Pathogen micro-organisms in samples taken at sampling points in milking and cheese manufacturing In this experiment the presence of pathogen micro-organisms Salmonella spp., Listeria monocytogenes, Escherichia coli, Campylobacter spp., sulphite-reducing clostridia and coagulase-positive staphylococci (Staphylococcus aureus and related species) was established in samples of udder surfaces, milking-machine surfaces, raw and mixed milk, whey after curdling, as well as cheese and brine. Listeria spp. was isolated from 7% of samples of four cows’ udder surfaces, one swab from milking machine inner surfaces, two milk and one whey samples, while none of the samples examined were positive to the presence of Salmonella spp. and Campylobacter spp. L. monocytogenes is a food-borne pathogen that can contaminate dairy products (Menendez et al., 2001). Listeria monocytogenes is in contrast to Salmonella a psychrotrophic microorganism and can survive at low temperatures. The growth of this organism on contaminated cheese can occur. The most commonly occurring species in food are L. innocua and L. monocytogenes. Outbreaks of listeriosis resulting from the consumption of dairy foods contaminated with L. monocytogenes have prompted concern about the behaviour of this organism during processing and the subsequent storage of various dairy products and about control of the hazard the bacterium poses to the dairy industry. Although Listeria is inactivated under normal conditions of pasteurisation (Schaack and Marth, 1988), problems can arise from post-pasteurisation contamination. Bacteria 54 Acta agriculturae slovenica, 84(december 2004)1. can enter cheese at many stages during its processing. Meyer Broseta et al. (2003) reported that the presence of L. monocytogenes in farm raw milk was low (only 2.4% of samples taken monthly from milk tankers). A seasonal effect (with peaks in winter) was observed. The farm milk contamination is, most often, a sporadic event. The number of bacterial cells of Listeria was also very low (below 3 bacteria per millilitre with a most probable concentration of 0.1 cfu/ml). Such low levels are very likely to be due to environmental contamination. 4 3.5 3 2.5 2 1.5 1 0.5 0 minimum average, povprečje maximum swabs before swabs after raw milk, mixed milk, whey, sirotka milking, brisi milking, brisi surovo mleko mešano pred molžo po molži mleko sampling points vzorčna mesta brine cheese, sir Figure 6. The average number (in log) of coagulase-positive staphylococci (S. aureus) in 98 samples taken at different sampling points of milking and cheese manufacturing (the results of swabs taken before and after milking are expressed in number of colony forming units cfu per 1 cm2 of milking machine surfaces and in other samples as number of cfu per 1 ml or 1 g). Slika 6. Povprečno število (v log) koagulaza-pozitivnih stafilokokov v 98 vzorcih, odvzetih na različnih kontrolnih točkah molže in sirjenja (brisi površine molznih strojev pred in po molži so izraženi kot število kolonijskih enot ke na 1 cm2, a pri ostalih vzorcih kot število ke na 1 ml ali 1 g). The contamination of raw milk with Salmonella usually occurs as a result of the transfer of faeces from an animal to milk via unclean teats and udders. Such contamination can pass into milk during milking and, once present, on milking parlour equipment that can then readily proliferate and spread if such equipment is not adequately cleaned and sanitised. Its growth in milk should be limited by effective refrigeration (<8 °C). Effective milking parlour hygiene (cleaning and disinfection of udders and teats), cleaning and sanitisation of milking equipment and subsequent milk storage systems are essential elements in preventing the spread of this organism (McManus and Lanier, 1987). Campylobacter jejunii and Campylobacter coli, which cause Campylobacter enteritis, may be commonly isolated from cow faeces and this is considered to be the main source of infection of raw milk. Campylobacter species do not generally grow at temperatures below 30 °C and are sensitive to the conditions necessary for growth. Therefore, growth is unlikely to occur in milk and dairy products. The infectious dose for these micro-organisms is, however, low and consequently growth may not be a prerequisite of infection. In raw milk, the Campylobacter Godič Torkar, K. and Golc Teger, S. Microbiological quality of some critical control points … cheese-makers. 55 number will normally be reduced during cold storage. Both Campylobacter species are sensitive to milk pasteurisation (Anonim., 1994). Campylobacter 0% Listeria sp. 7% S.aureus 57% E.coli 18% % of contaminated samples % okuženih vzorcev Clostridia 11% Figure 7. The percentage (%) of samples contaminated by pathogen micro-organisms of all 98 samples tested. Slika 7. Odstotek (%) s patogenimi mikroorganizmi kontaminiranih od skupno 98 preizkušenih vzorcev. Proteus was present in 7 (7%) cases of milk and whey. Habeš (2002) reported similar results. In his study Proteus spp. was present in 7.28% of 840 raw and pasteurised milk samples taken in Bosnia in a 4-year period. Sulfite-reducing clostridia (mostly Cl. perfringens) were detected in 10 (10%) samples (swabs, raw milk, whey). These spore-forms are present in sediment of various types, and in the intestinal tracts of men and animals. They gain entry to milk via faeces, soil and feedstuff, especially silage. Strains may be psychrotrophic, mesophilic or thermophilic. Since most strains are strictly anaerobic, they have the greatest potential importance as spoilage organisms of cheese and canned milk products. They produce a number of soluble toxic substances (Gilmour and Rowe, 1990). Escherichia coli was isolated in our experiment from 12 (12%) samples of swabs, raw and mixed milk, whey and brine. Testing for E. coli as an indicator of faecal contamination and/or poor hygienic practices has traditionally been done in dairy industry. It is well known that some strains may be enteropathogenic or enterotoxigenic. Both of these groups have been responsible for outbreaks involving cheese and milk (Anonim., 1994). The number of coagulase-positive staphylococci (Staphylococcus aureus and related species) exceeded the norms of the European Communities (M=2 000 bacteria per ml of sample) in 14.4% of raw milk samples. The average number of these organisms in raw milk was 5.7·102 and increased to 3.5·103 in whey samples (Fig. 6). The presence of coagulase-positive staphylococci was examined in 57% of samples (raw milk, mixed milk, whey, brine, cheese samples) (Fig. 7). De Buysier et al. (2001) reported that Staphylococcus aureus was by far the most frequent pathogen associated with food pathogen outbreaks (85.5% of the outbreaks) in France, followed by Salmonella (10.1%), E. coli (3%), L. monocytogenes (3%) and C. jejuni (1.5%) outbreaks. 56 Acta agriculturae slovenica, 84(december 2004)1. Coagulase-positive staphylococci (Staphylococcus aureus and related species) may cause human disease through the production of toxins. The formation of effective levels of toxin requires a high number of micro-organisms (approximately 105–106 micro-organisms per gram of food) at a pH value greater than 5 and so the presence of coagulase-positive staphylococci at a low level does not necessarily constitute a hazard. Dairy-related outbreaks of staphylococcal intoxication have been attributed to raw milk, dried milk, cheese and ice cream. Coagulase-positive staphylococci may be present in raw milk from the udder and teat canals of a cow, particularly if lesions are present. Also, the nasal area and hands of humans are recognised sites of contamination: poor personal hygiene can result in the contamination of milk and dairy products. Essential to the production of toxin is the growth of micro-organisms. In general, Staphylococcus aureus and the related species Staphylococcus intermedius and Staphylococcus hyius do not multiply at temperatures below 8 °C, and 10 °C is the minimum for toxin production. These micro-organisms are, however, resistant to salt. Pasteurisation will be effective against them but, if toxins are present, the toxins will not be inactivated. Therefore, toxins may be present in the absence of viable micro-organisms. The higher counts of Staphylococcus recorded in spring, when milk yields are at their peak, are a cause for concern and mammary infections (Anonim., 1994) (Fig. 7). Pathogen micro-organisms in cheese samples Salmonella, Listeria spp., Proteus, sulphite-reducing clostridia, Campylobacter were not detected in cheese samples. E. coli was found in 4 (30%) of samples on levels from 10–3400 cfu g–1 of cheese, while the coagulase positive staphylococci (S. aureus and related species) were present in 9 (64%) of samples and ranged from 100 to 50 000 cfu g–1 of cheese. Their average number in cheese samples (6.5·103) was in the same log range as in the whey samples (Fig. 6). These results showed a high contamination with these two types of micro-organisms in comparison with the results of Menendez et al. (2001) who established low average numbers of Staphylococcus aureus (<61 per g/cheese) and Escherichia coli (<52 per g/cheese) in 24 Tetilla cheese samples. Listeria monocytogenes was detected in two of 24 samples. None of the samples yielded Salmonella spp. A significant correlation (correlation coefficient r>0.80, P<0.0001) between the total bacterial count, the number of lactic-acid, non-lactic-acid-producers, enterococci, lactococci and lactobacilli in swabs, milk and cheese samples was established (data not shown). There were no significant differences in the number of micro-organisms between spring and autumn seasons, except for enterococci (P=0.0004**). Significant differences between the microbiological quality of samples from individual cheese-makers were also established (P=<0.0001***). There is no statistically significant influence on microbiological quality between different Slovenian areas where cheese production takes place, using starter cultures in cheese production or not, and between types of feeding (pasture, feeding in a cowshed, etc). Highly statistically significant differences in number of enterococci between cheese producers (P=<0.0001***) and between seasons (P=0.0041**) were found. There were also statistically significant differences between the number of bacteria E. coli between seasons (P=0.041), while the differences in E. coli numbers between producers were not statistically significant (P=0.36). Water samples Water used in the process of milk production should be of bacteriologically potable quality. The purity of properly treated supplies taken direct from the mains is assured, but bacterial contamination can be introduced from storage tanks not properly protected against rodents, birds, insects and dust. Bacteria may also come from dirty wash troughs, or the carrying of buckets and hoses. Many farms rely on untreated water supplies from boreholes, wells, lakes, springs and rivers; some of these may be contaminated at source with micro-organisms of faecal origin, e.g. Godič Torkar, K. and Golc Teger, S. Microbiological quality of some critical control points … cheese-makers. 57 coliforms, faecal streptococci and clostridia. In addition, a wide variety of saprophytic microorganisms derived from soil or from vegetation may be present, including Pseudomonas spp., coliforms and other Gram-negative rods, Bacillus spores, coryneform bacteria and lactic acid bacteria. The numbers of these contaminants vary widely. If untreated water gains access to milk or is used for rinsing equipment and containers, any micro-organisms present in the water will contaminate the milk although the numbers of micro-organisms added may not be significant in terms of the cfu/ml of milk. However, multiplication of some of the water-borne bacteria in any residual water in the equipment will result in a more serious contamination and may lead to the establishment and development of some undesirable types of micro-organisms, e.g. psychrotrophic Gram-negative rods, in the milking equipment. For these reasons, in areas where farm water supplies are bacteriologically unsatisfactory the chemical disinfection or sanitation of milking equipment is always delayed until just before the next milking, and the disinfectant solution is merely drained from the equipment before it is used for milking. This practice prevents recontamination resulting from rinsing with untreated water. Chlorination, by dosing with hypoclorite, is frequently recommended for water of unsatisfactory bacteriological quality used for the final rinsing of equipment, because it helps to reduce the risk of bacterial multiplication in residual water left in milking machines that are cleaned and sanitised in the one operation (Cousins et al., 1981). sulphite-reducing clostridia, sulfit-red. klostridiji Int. enterococci, Intest. enterokoki TBC(37), SŠ (37) TBC(25), SŠ (25) conforms, koliformni m.o. E.coli 0 10 20 30 40 50 60 70 % of opposable water samples % oporečnih vzorcev vode Figure 8. The percentage (%) of water samples not corresponding to the applicable regulations due to the presence or higher number of indicator micro-organisms: TBC (37): total bacterial count inc. at 37 °C; TBC (25): total bacterial count inc. at 25 °C, sulfite-reducing clostridia, coliforms, intestinal enterococci and E. coli. Slika 8. Odstotek (%) vzorcev vode, ki niso ustrezali veljavnim predpisom zaradi povišanega števila indikatorskih mikroorganizmov: SŠ (37): skupno število m.o. ob inkubaciji pri 37 °C; SŠ (25): skupno število m.o. ob inkubaciji pri 25 °C, sulfit-reducirajočih klostridijev, koliformnih m.o., intestinalnih enterokokov in E. coli. The number of total bacterial count and the presence of E. coli, coliforms and faecal streptococci in water samples taken at taps or water pipes in cheese-makers and in milking parlours were established in our study. The results showed that 78% of water samples did not correspond to the microbiological criteria according to the applicable regulations (Pravilnik, Ur. 58 Acta agriculturae slovenica, 84(december 2004)1. l. RS, 2004). E. coli was present in 22% of samples, coliforms in 67% and faecal enterococci in 22% of the samples. The number of viable micro-organisms at 37 °C was exceeded in 11% of samples, while the number of micro-organisms at 22 °C was exceeded in 33% of samples (Fig. 8). System of critical control points In recent years, the hazard analysis critical control points (HACCP) concept has been proposed as the best approach to ensure food safety. The results of this study also underline the need to achieve food safety and reduce risk, to implement the hazard analysis critical control points (HACCP) concept and quality assurance from the farm to the dairy plant, and to set up and apply EU directive 92/46 on milk hygiene (Silva, 2003; De Buyser, 2001). It is very important for cheese-makers to set up the system of critical control points and to investigate the direct and cross-contamination sources in their cheese production. The milking machines, production pipelines, equipment such as vats, plastic wraps, pressing cloths, starter cultures and the hands of workers were direct contamination sources. In addition, the hands of workers and the water used in a facility played a role in direct cross-contamination. The air in the facility was a critical control point for yeast and mould contamination. Routine microbiological monitoring of the hygienic quality of raw milk should be employed using not only the total plate count, but also indicator bacteria such as E. coli or coliforms and incentive payment schemes should be considered where milk is intended to be used without a bacterial destruction stage in the process, i.e. for raw milk cheese, to encourage the adoption of high hygienic standards (McManus and Lanier, 1987). The inclusion of other pathogen microorganisms like L. monocytogenes on the list of organisms subject to the HACCP has recently also been called for (Silva et al., 2003). CONCLUSIONS - In our study 21% of tested cheese samples did not correspond to the microbiological criteria according to the applicable EU and Slovenian regulations. - The high number of micro-organisms on the surfaces of washed milking machines before milking showed ineffective cleaning (washing) by about 60% of cheese producers. - Greater contamination usually appeared during cheese-processing and not during milking. - In 78% of drinking water samples the results exceeded the microbiological criteria according to the applicable regulations so greater attention should also be paid to water quality. - It is suggested that more importance should be given to milking and cheese production hygiene, as well as to the determination and control of critical points in firms for improving cheese quality and preventing food-borne pathogenic outbreaks. - The authors believe that milk and cheese producers should employ the HACCP and quality assurance practices in the production stages of milk from the farm up to and including the dairy plant, while also setting up and implementing EU Directive 92/46. POVZETEK V Sloveniji je posebno v odročnejših predelih kar nekaj individualnih majhnih sirarn, kjer proizvajalci mleko sami predelajo v sire, včasih tudi v skuto. Pogosto uporabljajo za sirjenje surovo, nepasterizirano mleko, kar omogoči boljši izkoristek mleka, ohrani pa se tudi naravna, za tisto področje značilna mlečnokislinska mikroflora, ki igra pomembno vlogo pri senzoričnih Godič Torkar, K. and Golc Teger, S. Microbiological quality of some critical control points … cheese-makers. 59 značilnostih proizvedenih sirov. Higienska kakovost in zdravstvena ustreznost proizvedenih sirov zavisi od ustrezne mikrobiološke kakovosti mleka kot surovine, razmer, v katerih se mleko predeluje v sire, kakovosti molže, temperature hranjenja mleka in sirov, krme, sezone, uporabe različnih starterskih kultur, vode, uporabljene za napajanje, pranje molznega in mlekarskega pribora itd. Preverjanje prisotnosti in števila specifičnih mikroorganizmov v različnih fazah prireje mleka in njegove predelave je pomemben dejavnik pri kontroli in sistemu zagotavljanja kakovosti proizvodnje. Namen našega dela je bil ugotoviti mikrobiološko kakovost, oziroma prisotnost posameznih skupin mikroorganizmov na nekaterih kritičnih kontrolnih točkah prireje mleka in sirjenja. V ta namen smo ugotavljali prisotnost patogenih in indikatorskih mikroorganizmov v 14 vzorcih poltrdega sira, proizvedenih pri posameznih sirarjih na različnih področjih Slovenije ter v 98 vzorcih, odvzetih v postopku molže in predelave mleka v sir. Odvzeli smo brise površine vimena krav molznic ter notranjih površin molznih strojev, vzorce surovega mleka takoj po molži, mešanega mleka iz sirarskega kotla, sirotke po usirjanju, slanice, sirov po enomesečnem zorenju in vakuumsko pakiranih sirov po naknadnem enomesečnem skladiščenju pri temperaturi 6 °C. Odvzeli smo vzorce vode, namenjene pranju molznega in mlekarskega pribora. Ugotovili smo, da 3 (21 %) vzorci sirov glede mikrobiološke kakovosti niso ustrezali kriterijem slovenske zakonodaje. Visoko število mikroorganizmov na notranjih površinah molznih strojev (kolektorji, sesne gume) pred molžo kažejo na neučinkovitost pranja (čiščenja, dezinfekcije) pri kar 60 % proizvajalcev. V postopku predelave mleka v sir je prišlo pogosto do večje okužbe kot v postopku molže. Kar 78 % vzorcev vode glede mikrobiološke kakovosti ni ustrezalo predpisanim kriterijem, zato je potrebno veliko pozornost usmeriti na problem zagotavljanja kakovosti vode v odročnejših predelih. Prav tako predlagamo, da posamezni sirarji posvetijo večjo pozornost izboljšanju higiene pri molži in sirjenju ter se tako izognejo slabi mikrobiološki in senzorični kakovosti ter zdravstveni oporečnosti svojih proizvodov. Vzpostavitev sistema kontrole kritičnih točk v posameznih stopnjah prireje in predelave mleka v smislu sistema HACCP in zagotavljanja kakovosti je zaželena in tudi zakonsko predpisana z direktivami EU. ACKNOWLEDGEMENT This work was supported by Ministry of Education, Science and Sport, and by Ministry of Agriculture, Forestry and Food of the Republic of Slovenia. REFERENCES Angelotti, R./ Hall, H.E./ Foter, M.J./ Lewis, K.M. Quantitation of Clostridium perfringens in Foods. Appl. Microbiol., 10(1962), 193–199. Anonimus. Recommendations for the hygienic manufacture of milk and milk based products, appendix A.1 Spoilage and pathogenic bacteria in milk based products. Bulletin of the International Dairy Federation, IDF 292/1994, Brussels, Belgium, (1994), 28–32. Anonimus. CCFRA Microbiological Methods Manual. Compendium of Microbiological Methods for the Analysis of Food and Agricultural Products, Mesophilic Spore Count, Method 2.3.: 1995, 2nd Edition 2002, the Scientific Association dedicated to Analytical Excelence, Campden, UK, http://127.0.0.1:8080//data/ccfra/2_3/2_3.htm, (2002), 1–3. Arenas, R./ Gonzales, L./ Bernardo, A./ Fresno, J.M./ Tornadijo, M.E. Microbiological and physico-chemical changes in Genestoso cheese, a Spanish acid curd variety, throughout ripening. Food Control, 15(2004)4, 271–279. Bramley, A.J./ McKinnon, C.H. The Microbiology of Raw Milk. In: Dairy Microbiology, I, (Ed.: Robinson, R. K.). London, New York, Elsevier Applied Science, 1990, 171. 60 Acta agriculturae slovenica, 84(december 2004)1. Cousins, C.M./ Bramley, A.J. The Microbiology of Milk. In: Dairy Microbiology, I. (Ed.: Robinson, R. K.). London, New York, Elsevier Applied Science, 1990, 142–143. De Buyser, M.L./ Dufour, B./ Maire, M./ Lafarge, V. Implication of milk and milk products in food-borne diseases in France and in different industrialised countries. J. Food Microbiol., 67(2001)1–2, 1–17. De Man, J.D./ Rogosa, M.A./ Sharpe, M.E. Medium for the cultivation of lactobacilli. J. Appl. Bact., 23(1960)1, 130–135. EN ISO 11290-1(E). Microbiology of food and animal feedeing stuffs – Horizontal method for the detection and enumeration of Listeria monocytogenes. Part 1: Detection method (ISO 11290-1, 1996). International Organization for Standardization, Genova, Switzerland, (1996), 1–16. EN ISO 4833. Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of micro organisms-Colony-count technique at 30 °C. International Organization for Standardization, Genova, Switzerland, (2003), 1–9. Estepar, J./ Sanchez, del Mar, M./ Alonso, L./ Mayo, B. Bichemical and microbiological characterization of artisanal Penamellera cheese:analysis of its indigenous lactic acid bacteria. Int. Dairy J., 9(1999)10, 737–746. Evrensel, S.S./ Temelli, S./ Anar, S. Detection of critical control points in white cheese production in small dairy plants. Turk Veterinerlik ve Hayvancilik Dergisi, 27(2003)1, 29–35. FIL-IDF 149A. Dairy starter cultures of lactic acid bacteria. Standard of identity. International Dairy Federation, Brussels, Belgium, (1997), 1–8. Gelsomino, R./ Vancanneyt, M./ Cogan, T.M./ Condon, S./ Swings, J. Source of enterococci in a farmhouse raw milk cheese. Appl. Environm. Microbiol., 68(2002)7, 3560–3565. Gilmour, A./ Rowe, M. T. Micro-organisms Associated with Milk. In: Dairy Microbiology, I. (Ed.: Robinson, R. K.). London, New York, Elsevier Applied Science, 1990, 37–76. Habeš, S. Qualitative-quantitative analysis of micro-organisms biodiversity in raw and pasteurised milk. Mljekarstvo, 52(2002)4, 291–313. ISO 9308-2 (E). Water quality-Detection and enumeration of coliform organisms, thermotolerant coliform organisms and presumptive Escherichia coli-Part 2: Multiple tube (most probable number) method. International Organization for Standardization, Genova, Switzerland, (1990), 1– 9. ISO 5667-2. Water quality-Sampling-Part 2: Guidance on sampling techniques (ISO 5667-2, 1991), International Organization for Standardization, Genova, Switzerland, (1991), 1–9. ISO 6611 (E). Milk and milk products-Enumeration of colony-forming units of yeasts and/or moulds – Colony-count technique at 25 °C, International Organization for Standardization, Genova, Switzerland, (1992), 1–6. ISO 10272 (E). Microbiology of food and animal feeding stuffs – Horizontal method for the detection of thermotolerant Campylobacter. International Organization for Standardization, Genova, Switzerland, (1995), 1–15. ISO/DIS 707. Milk and milk products-Guidence on sampling. International Organization for Standardization, Genova, Switzerland, (1995), 1–41. ISO 6888-2. Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) Part 2: Technique using rabbit plasma fibrinogen agar medium. International Organization for Standardization, Genova, Switzerland, (1999), 1–7. ISO/DIS 5541-1. Milk and milk products-Enumeration of coliforms-Part 1: Colony count technique at 30 °C without resuscitation. International Organization for Standardization, Genova, Switzerland, (1999), 1–7. ISO/FDIS 8261 (E). Milk and milk products – General guidance for the preparation of test samples, initial suspensions and decimal dilutions for microbiological examination. International Organization for Standardization, Genova, Switzerland, (2001), 1–12. ISO 6579. Microbiology of food and animal feeding stuffs – Horizontal method for the detection of Salmonella spp. (ISO 6579:2002). International Organization for Standardization, Genova, Switzerland, (2002), 1– 27. McManus C./ Lanier, J.M. Salmonella, Campylobacter jejuni and Yersinia enterocolitica in raw milk. J. Food Prot., 50(1987)1, 51–55. Menendez, S./ Godinez, R./ Centeno, J.A./ Rodriguez-Otero, J.L. Microbiological, chemical and bichemical characteristics of « Tetilla »raw cows-milk cheese. Food Microbiol., 18(2001)2, 151–158. Methodenbuch M 7.16.2. Milch und Milchprodukte, Säurebildner, Bestimmung säurebildner Mikroorganismen, M 7.16.2, Methodenbuch, Band IV, VDLUFA, Verlag Darmstadt, Germany, (1985), 2. Methodenbuch M 7.3.3. Milch und Milchprodukte, Eiweisszersetzer, Bestimmung von Eiweisszersetzern (Proteolyten), M 7.3.3, Methodenbuch, Band IV, VDLUFA, Verlag Darmstadt, Germany, (1985), 2. Methodenbuch M 7.6. Milch und Milchprodukte, Fettspalter, Bestimmung von Fettspaltern (Lipolyten), M 7.6, Methodenbuch, Band IV, VDLUFA, Verlag Darmstadt, Germany, (1985), 2. Methodenbuch M 7.18.3.1. Milch und Milchprodukte, Sporenbilder, anaerob, Bestimmung von käsereischädlichen Clostridien, M 7.18.3.1, Methodenbuch, Band IV, VDLUFA, Verlag Darmstadt, Germany, (1995), 4. Meyer Broseta, S./ Diot, A./ Bastian, S./ Riviere, J./ Cerf, O. Estimation of low bacterial concentration: Listeria monocytogenes in raw milk. Int. J. Food Microbiol., 80(2003.)1, 1–15. Godič Torkar, K. and Golc Teger, S. Microbiological quality of some critical control points … cheese-makers. 61 Officional Journal of the European Communities.Council directive 92/46/EEC, of 16 June 1992 laying down the health rules for the production and placing on the market of raw milk, heat-treated milk and milk-based products, ANEX A: Requirements relating to the acceptance of raw milk at treatment and/or processing establishments, ANEX C, Chapter II: Microbiological criteria for milk-based products and drinking milk No L 268(1992), 1–19, 26–28. Pravilnik o načinu opravljanja veterinarsko sanitarnega pregleda in kontrole živali pred zakolom in proizvodov živalskega izvora. Ur. l. SFRJ, št. 68(1989), 1698–1699. Pravilnik o pitni vodi. Ur. L. RS št.19(2004), 2155–2166. Pravilnik o veterinarsko-sanitarnem nadzoru živilskih obratov, veterinarsko-sanitarnih pregledih ter o pogojih zdravstvene ustreznosti živil in surovin živalskega izvora. Uradni list RS št. 28(2004), 3183–3197. Salmeron, J./ De Vega, C./ Perez-Elortondo, F.J./ Albisu, M./ Barron, L.J.R. Effect of pasteurization and seasonal variations in the microflora of ewe’s milk for cheesemaking. Food Microbiol., 19(2002)2–3, 167–174. Silva, I. M.M./ Almeida, R.C.C./ Alves, M.A.O./ Almeida, P.F. Occurence of Listeria spp. in critical control points and the environment of Minas Frescal cheese processing, Int. J. Food Microbiol., 81( 2003)3, 241–248. SIST EN ISO 6222. Water quality - Enumeration of culturable micro-organisms-Colony count by inoculation in a nutrient agar culture medium. International Organization for Standardization, Genova, Switzerland, (1999), 1–2. SIST EN ISO 7899-2. Water quality-Detection and enumeration of intestinal enterococci-Part 2: Membrane filtration method (ISO 7899-2:2000). International Organization for Standardization, Genova, Switzerland, (2000), 1–7. Terzaghi, B.E./ Sandine, W.E. Improved medium for lactic streptococci and their bacteriofages. Appl. Microbiol., (1975), 807–813. Valjavec, I. Slovene dairies. Cattle Bells, Periodical of Cattle Breeders’ Association of Slovenia, 5(2000)1–2, 39–43. Valjavec, I. Odkup in predelava mleka do leta 2002 v slovenskih mlekarnah. Seminar: Mleko in sadni sokovi, Hmezad Export Import Inženiring, Lipica, maj, 2003. Acta agriculturae slovenica, 84(december 2004)1, 63-80. http://www.bfro.uni-lj.si/zoo/publikacije/zbornik Agris category codes: E20 COBISS Code 1.02 THE CONSEQUENCES OF CHANGING CONDITIONS OF THE EUROPEAN DAIRY SECTOR FOR THE STRATEGIES OF DAIRY COMPANIES Hannes WEINDLMAIER a) a) Technical Univ. of Munich, Professor for Dairy and Food Industry Management, Weihenstephaner Berg 1, D-85350 Freising, Prof., Ph.D., E-mail: h.weindlmaier@wzw.tum.de. Received June 10, 2004, accepted October 29, 2004. Delo je prispelo 10. junija 2004, sprejeto 29. oktobra 2004. ABSTRACT This contribution deals with the consequences of changing conditions of the European dairy sector. Important influences discussed are the decisions of the Mid-Term-Review of the Common Agricultural Policy, the enlargement of the European Union to include the countries of Central and Eastern Europe and the concentration and globalisation of the food trade. Based on these changes, the dairy industry has to adjust their strategies. Of great importance for future competitiveness is the size of dairy companies and plants, as large enterprises and plants have several economic advantages, if an optimal size corresponds with a good job performed by the management of the company. Important alternative strategies like the cost leadership strategy, the differentiation strategy, the focus strategy and internationalisation are discussed and assessed with respect to their possible contribution to retain respectively to improve competitiveness of dairy enterprises. It is concluded that within a period of ten years the face of the European dairy sector will change quite extensively. Only those enterprises will survive which in time actively implement strategies, which are a suitable answer to the changing conditions. Key words: milk production / dairy sector / conditions / EU enlargement / globalisation / strategies / competitivity IZVLEČEK Prispevek analizira posledice spremenjenih pogojev, v katerih deluje evropska mlekarstvo. Obravnava pomemben vpliv odločitev srednjeročnega poročila o Skupni kmetijski politiki, širitev Evropske Skupnosti z vključitvijo držav Srednje in Vzhodne Evrope ter koncentracijo in globalizacijo trgovine. Upoštevaje te spremembe mora mlekarska industrija prilagoditi svoje strategije. Velikega pomena za konkurenčnost v prihodnosti je velikost mlekarskih firm in obratov, ker imajo ob dobrem upravljanju veliki obrati in podjetja številne prednosti. Podan je pregled pomembnih alternativnih strategij, kot so strategija cene vodenja, strategija diferenciacije, strategija fokusiranja in strategija internacionalizacije ter diskusija možnega doprinosa teh strategij k večji konkurenčnosti mlekarskih podjetij. Podana je napoved, da se bo stanje v evropski mlekarski industriji v naslednjih desetih letih močno spremenilo. Preživela bodo samo tista podjetja, ki bodo aktivno in pravočasno uveljavila strategije, ki pomenijo ustrezen odgovor na spremenjene pogoje. Ključne besede: mlekarstvo / pogoji / širitev EU / globalizacija / strategije / konkurenčnost INTRODUCTION In order to analyse the future competitiveness of the dairy sector Porters diamond is most helpful to be used as a framework (Porter, 1998, Pitts and Lagnevik, 1998, Weindlmaier, 2000). This approach identifies six sources of comparative advantage, i.e. the factor conditions, firm 64 Acta agriculturae slovenica, 84(december 2004)1. strategy, structure and rivalry, demand conditions, related and supporting industries, government and chance. In this contribution, the analysis is limited to changes in the economic environment of the European dairy sector which are of specific current relevance. First of all, the changes in the Common Agricultural Policy according to the decisions of the Mid Term Review (MTR) of June 26th, 2003 are discussed. Then the enlargement of the European Union to include the countries of Central and Eastern Europe and developments in food retail trade are presented and analysed. The second part discusses the main strategic options which are at the disposal of the dairy industry as a reaction to these changes. In this connection, the main emphasis is given to structural changes in the dairy processing sector. THE CHANGING CONDITIONS FOR THE EU DAIRY INDUSTRY Forthcoming changes in the common market order for the dairy sector and its implications With respect to the changing policy environment affecting the dairy industry the decisions of the MTR of the Common Agricultural Policy of June 26th, 2003 have to be taken into account. Referring to the dairy sector, the following decisions are of specific importance: First of all, the dairy quota system was prolonged until 2014/15. The start of the general quota increase of 1.5% decided within the Agenda 2000 was postponed until 2006 (0.5% per year). Secondly, starting in 2004, the intervention prices of butter will be cut by 25% (3×7% and 1×4%) and the intervention price of skimmed milk powder by 15% (over three years, 5% each). The third important decision refers to limitations of the intervention of butter: Intervention purchases of butter will be suspended above a limit of 70,000 tons per year in 2005/06. This amount will be reduced by further 10,000 tons per year until 2008/09 (30,000 tons thereafter). Furthermore, butter intervention will be restricted to the period March 1st to September 1st. A new element for the EU dairy policy is the introduction of direct payments to at least partly compensate the price reduction of intervention prices. Direct payments to dairy farmers will be introduced, starting in 2004/05 with 1.18 Cent/kg and increasing to 3.55 Cent/kg in 2006/07 and after. In connection with the introduction of direct payments it has been decided that these so called Single Farm Payments will be decoupled from the volume and kind of production. This decoupling is expected to reduce output by EU dairy farmers. However, member states may choose to maintain a limited link between subsidy and production under well defined conditions and within clear limits. Furthermore, the principle of Cross Compliance has been introduced, which means that direct payments are conditional on compliance of production with environmental, food safety, animal welfare and occupational safety standards. The implications of the MTR for milk producers (for milk production) For milk producers because of falling intervention prices and the extension of quotas a significant drop in producer milk prices is expected. Fig. 1 shows that over time the producer milk prices followed quite closely the support prices by intervention. Therefore, if these support prices decrease from about 28 Cent/kg in 2003 to 22 Cent/kg in 2007, a significant drop in producer milk prices is likely. This drop in producer milk prices will only be partly compensated by direct payments to the farmers. Weindlmaier, H. The consequences of changing conditions of the European dairy sector … dairy companies. 65 Cent kg-1 34 32 30 28 26 24 22 20 30.53 31.40 29.18 / \ 29.50 28.58 28.06 28.06 28.06 28.06 28.06 28.06 28.06 28.06 28.06^ 26.19 24.32 24.45 21.92 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 -Average EU producer milk price - Support price by intervention Source: Own presentation based on ZMP Zentrale Markt- und Preisberichtsstelle, 2003 Figure 1. Development of the average producer milk prices in the EU and of the EU support price by intervention. Slika 1. Razvoj povprečnih stroškov proizvajalcev mleka v ES in podpornih cen z intervencijami ES. Source: IFCN International Farm Comparison Network, 2003 Figure 2. Costs of Milk Production and Producer Milk Price. Slika 2. Cena proizvodnje mleka in cena mleka. 66 Acta agriculturae slovenica, 84(december 2004)1. A second outcome for dairy farmers will be the fact that a comprehensive coverage of the costs of milk production by the returns received for milk will further decline. Subsequently, the consequences are most probably significant decreases in farmer incomes. Fig. 2 indicates that already in 2002 in some European countries the total costs of milk production are not covered entirely by the returns received for milk. Therefore, if prices will decline as shown, this coverage of full economic costs will further decrease. In this figure, the opportunity costs refer to costs for own production factors inside the enterprise (own land, family labour, own capital), the abbreviations below the bars refer to farm codes, i.e. DE-35 means a German 35-cow farm. From this, the third consequence is quite likely: In regions with high production costs and low competitiveness for milk production, like for instance in mountainous regions of Southern Germany, France and Austria, milk production will be significantly reduced in the long run. This furthermore means that a universal coverage of grassland management, which has been an objective for agricultural policy for a long time, cannot be continued. The implications of the MTR for the dairies If the MTR is realised, dairies potentially have an advantage because of the fact that producer milk prices will decrease. Consequently, the production costs of the dairies will decrease, too. A growing international competitiveness could be the outcome. This indeed is one of the most important aims of the EU commission for changing Common Agricultural Policy for the dairy sector. However, in regions with a low profitability of milk production, e.g. if small farms are prevailing, the density of milk production will probably be cut dramatically as a consequence, leading to a corresponding loss of raw milk for the dairies. A relocation of dairy plants connected with the need for regional disinvestment programs will probably be the logical consequence. As an effect of the decisions of the MTR, the need for structural change in the dairy sector will further be intensified. The accession of the Central and Eastern European Countries to the European Union The European Union realised its biggest enlargement ever in terms of scope and diversity. 13 countries have applied to become new members. Ten of these countries including Slovenia joined the EU on May 1st, 2004. In order to join the EU, the new countries need to fulfill the economic and political conditions known as the “Copenhagen criteria”. The dairy sector of the new member states will have full and immediate access to CAP market measures, such as export refunds and skimmed milk powder and butter intervention, which will contribute to stabilising the markets. Of great importance are the decisions concerning the milk quotas for the new member states in relation to the consumption expectations and the decision concerning the quality of products and the hygienic standards (table 1). The table shows that the CEEC accession countries got a milk quota of 18.3 million tons. This quota can be extended by a reserve to a maximum of 19.0 million tons in 2006. If this amount is compared with the estimates concerning the demand of dairy products, this decision means that there is a potential need for imports of 1.2 million tons in 2004.1 Of similar importance are the decisions concerning quality and hygienic standards, as with the admission into the EU all EU-quality and hygienic standards are relevant and have to be fulfilled. Exports to EU- and other countries are only allowed for processors with an export license. In most of the CEEC-countries intensive activities are performed to increase quality standards and to get export licenses. However, from the experience Germany made with the integration of the German Democratic Republic we know that this is a rather long and expensive process. This Weindlmaier, H. The consequences of changing conditions of the European dairy sector … dairy companies. 67 is especially true for Poland, the largest milk producing country of the accession countries. In 2003, out of the 330 dairies in Poland only 49 had an export licence to EU-countries (Pieniadz, 2004). Table 1. Relevant decisions concerning EU-extension to the CEEC-countries Preglednica 1. Pomembne odločitve povezane s širitvijo ES (1) Decisions concerning milk quotas Milk quota after 2004 18.3 million tons Milk quota inclusive reserve after 2006 19.0 million. tons Actual dairy production in access countries 21.5 million tons Estimated demand of the CEEC-countries 19.5 million tons Potential need for imports 2004 1.2 million tons (2) Decisions concerning milk quality ¦> With the admission into the EU all rules concerning quality and hygienic standard are relevant -however only a small part of the dairies has licenses for exports. ¦> Special quality and hygienic arrangements until 2006, but for national markets only. Source: Weindlmaier, 2003 based on Richarts, 2003. From this situation several consequences for the dairies are expected. First of all, the degree of competition in the markets for dairy products in the CEEC countries will increase quite extensively. Dairy companies in the accession countries which are not able to adjust to the hygienic standards set by the EU and to increase the quality of their products very fast, will not be competitive in the common European market. It is expected, that many dairy companies in the accession countries will get out of business during the next few years. The EU enlargement will also have consequences for the trade flows within Europe. At the one hand it is likely that the CEEC countries will increase the export of basic dairy products like cheese and milk powder to the countries of Western Europe. In the adjacent regions to Western European countries even raw milk might be transported for processing in processing plants of the West. At the other hand it is quite likely that Western European dairy companies will increase the export of branded dairy products and specialities to the CEEC countries. In addition, the number of joint ventures and direct investments will further rise. Concentration and globalisation in the food trade During the last few decades significant changes took place within the global food trade. Table 2 illustrates first of all the enormous, border-spreading concentration processes in the food trade. The largest food trade company world-wide, Wal-Mart, has a yearly turnover of 243 billion Euros. In addition to this huge, US based enterprise with several affiliates in Europe, we note very large European companies like Carrefour, ITM Enterprises, and Auchan in France, Ahold in the Netherlands, Metro, Rewe, Aldi, Edeka, and Tengelmann in Germany, Tesco and Sainsbury in Great-Britain. Based on strong bargaining power, the food retail trade is more or less fixing purchase prices for the products delivered by the dairy industry. In addition, the food trade forces suppliers to 68 Acta agriculturae slovenica, 84(december 2004)1. accept increasing price deductions to support the advertising expenses of the food trade and to perform specific payments for including articles of the company into the assortment of outlets. Table 2. The Top-20 food trade companies world-wide in 2002 Preglednica 2. Vodilnih 20 prehranskih trgovskih podjetij na svetu v letu 2002 Company Country Turnover 2001, million EUR Share of food, % Foreign sales, % 1 Wal-Mart USA 243,281 40.0 18.0 2 Carrefour France 69,486 70.5 50.6 3 Ahold Netherlands 66,593 92.0 86.5 4 Kroger USA 55,959 92.0 86.5 5 Metro Germany/CG 49,522 49.7 44.0 6 Albertson’s USA 42,781 90.0 0.0 7 Kmart USA 38,655 37.0 0.0 8 Safeway USA 48,314 92.0 10.1 9 Costco USA 38,131 41.0 18.0 10 Tesco Great-Britain 38,059 90.0 15.0 11 Rewe Germany 37,540 70.3 20.5 12 Aldi Germany 32,400* 84.0 39.4 13 ITM Enterprises France 31,900* 82.4 25.5 14 J. Sainsbury Great-Britain 29,743 90.0 15.0 15 Ito-Yokado Japan 29,624 47.0 36.0 16 Edeka/AVA Germany 28,035* 84.0 8.6 17 Aeon (Jusco) Japan 26,680 44.0 11.8 18 Tengelmann Germany 25,670* 74.6 57.6 19 Auchan France 25,500* 70.0 35.0 20 Supervalu USA 23,243 76.0 0.0 * = Estimate; Source: M+M Planet retail, www.planetretail.net Table 2 shows that a high percentage of the turnover of these companies is not realised by outlets in the home country of the respective company but by their foreign subsidiaries. For example, Metro, the largest German food trade enterprise, has affiliates in 18 European countries. As a consequence of the national and international concentration processes only large suppliers are adequate partners for these food trade companies. Purchasing activities of these food trade giants are continually centralised which means that large quantities are needed for the great number of outlets. In the future, international sourcing will gain importance. A second important development refers to changes in retailing food products. The typical supermarket lost market shares while large self-service department stores and low-price discount stores quickly gained importance. In addition, the establishment and penetration of private labels has become a preferred sales strategy. The fast growing introduction of private labels by the food trade consequently forced even leading suppliers of brands to produce private labels in spite of the risks associated with such activities. Fig. 3 illustrates the fast growth of discount stores in different European countries. By far the highest percentage of discount stores we find in Germany, as shown in Fig. 4. Meanwhile more than 50% of dairy products are sold in stores of Aldi, Lidl, Penny, etc. This development is particularly important insofar as low prices represent important sales arguments of these discount stores. Intense price struggles are performed between some of the low-price Weindlmaier, H. The consequences of changing conditions of the European dairy sector … dairy companies. 69 chains. Important for the dairy industry is the fact that these competitors attempt to realise their low price strategy primarily by low purchasing prices of the products supplied by the dairies. 40 35 30 25 20 15 10 I J Q 1988 ¦ 1993 D 1998 ? 2002 Jlëj Austria Denmark France Source: Own presentation based on ACNielsen, 2003. land Great Italy Spain Switzer-Belgium Finland Germany Netherlands Britain Figure 3. Development of market share of discount stores in Europe. Slika 3. Razvoj tržnega deleža veletrgovin v Evropi. ´99 00 01 02 03 Jan-June Milk 1) exclusive drinking yoghurt ´99 00 01 02 03 Jan-June Yoghurt1) 46 46 ´99 00 01 02 03 Jan-June Butter Aldi Discount stores except Aldi ´99 00 01 02 Cheese Source: Own presentation based on ZMP-data derived from GfK-Panel of households. ZMP Zentrale Markt- und Preisberichtsstelle, Bonn 2003a 5 0 Figure 4. Percentage of dairy products sold in low price discount stores in Germany Slika 4. Odstotek mlečnih izdelkov prodanih v veletrgovinah znizkimi cenami v Nemčiji 70 Acta agriculturae slovenica, 84(december 2004)1. A third development is the ongoing reorganisation of the relationship between the food trade and the dairy industry. One aspect of this process is the establishment of ECR (Efficient Consumer Response) to optimise the supply chain. Dairy companies are asked to introduce such systems together with the food trade companies. Behind this development stands the demand for closer co-operation in the ranges product development, assortment optimisation, logistics and storage. Another aspect is a reaction of the diverse food scandals in the 90ies: To improve the safety of the food products sold, the food trade companies nowadays demand from the suppliers the implementation of quality management standards. Food safety systems of the manufacturers, e.g. IFS or BRC, are more or less presupposed and aspects of food security are becoming increasingly important (Schiefer, 2003; Weindlmaier, 2003). STRATEGIES OF THE DAIRY INDUSTRY TO IMPROVE COMPETITIVENESS UNDER THE GIVEN CONDITIONS Structural change and concentration in the dairy industry Past structural changes and concentration in the dairy industry on the one hand are an important issue for the present conditions of the dairy sector today. However, the growth of companies is on the other hand an important strategy to retain and improve competitiveness. Past developments of the dairy structure In general a strong decline in the number of companies and plants in the dairy industry in almost all countries of the Western World can be observed . Table 3 illustrates the structural change in the dairy industry of selected countries between 1983 and 1997 in terms of milk collection by dairy firms. During this period the number of companies decreased in all countries quite extensively, i.e. between 25% in Ireland and 52% in Germany. The right side of the table shows that at the same time the volume of milk collected increased by up to 156% in the United States. These data also illustrate that there are big differences concerning the size of dairies. While average milk collection in French companies has been 30.2 million litres only, in the Netherlands nearly 500 million litres have been collected per average company. Table 3. Structural change in the dairy industry of selected countries Preglednica 3. Strukturne spremembe v mlekarski industriji izbranih držav No. of fiems in milk collection Average volume collected, mio. lit. 1983 1997 % Change 1983 1997 % Change Germany 528 256 -52 57.3 106.0 85 France 1570 734 -50 15.5 30.2 95 Netherlands 42 21 -50 317.5 498.0 57 Ireland 51 39 -25 100.3 131.0 30 United States 1 435 226 -48 100.0 256.0 156 Canada 250 171 -32 28.9 43.4 50 United States data refer to 1980 rather than 1983 and relate only co-operatives. Source: Pitts and Krijger, 2001. Table 4 shows Europe’s major milk processors in 2001/2002 in terms of milk purchases per year. The largest companies like Arla Foods and Group Lactalis process more than seven billion litres per year, that means more than 15 times the milk deliveries of 461 million litres to all dairy Weindlmaier, H. The consequences of changing conditions of the European dairy sector … dairy companies. 71 companies in Slovenia in 2001 (ZMP, 2003). Out of the Top-15 companies about half are Coop’s and half are private dairy enterprises. From Fig. 5 we can derive that the largest dairy producers worldwide are at the one hand multinational companies like Nestlé, Danone, Kraft Foods and Unilever. Several very large companies are operating in the United States, but also in some of the European countries, in which the concentration processes progressed very far. Examples are the Scandinavian countries like Denmark and Sweden with the largest European dairy company Arla Foods or the Netherlands with Friesland Coberco and Campina. Table 4. Europe’s major milk processors in 2001/2002* Preglednica 4. Največji predelovalci mleka v Evropi v letih 2001/2002 Rank Company Co-op (C) or Private (P) Country of origin Milk purchases million lit./year 1 Arla Foods C Denmark/Sweden 7,200 2 Group Lactalis P France 7,000 3 Campina C Netherlands 5,750 4 Friesland Coberco C Netherlands 5,600 5 Nordmilch C Germany 4,200 6 Bongrain/CLE P France 4,100 7 Nestlé P Switzerland 2,800 8 Dairy Crest P UK 2,700 9 Humana Milchunion C Germany 2,460 10 Glanbia C/P Ireland 2,450 11 Danone P France 2,430 12 Sodiaal C France 2,300 13 Entremont P France 1,950 14 Müller P Germany 1,850 15 Laita Group C France 1,730 * = Source: Barry Wilson’s Dairy Industry Newsletter Online, 2003. Typical for the recent concentration processes is the fact that those are not only characterised by takeovers of small or medium sized companies by the large ones. In addition, mergers and takeovers of very big enterprises are at the agenda. Examples are the merger between MD foods and Arla in 2000 and the takeover of the English Express Dairies by Arla Foods and of the German dairy part of Unilever by the French giant Bongrain in 2003. Another characteristic of recent concentration processes is the fact of transnational mergers, acquisitions, joint ventures, takeovers and strategic alliances. Recent examples of transnational alliances are the transnational strategic alliance between the French Lactalis and the Danish Tholstrup Cheese in 2001 and the alliance between Nestlé and the New Zealand Dairy Group (Fonterra) for the American market in 2002. If these developments in concentration are analysed, the question about the future development of concentration arises. There are indeed no signs that the speed of these concentration processes have diminished in recent years. For 2000 to 2003 the Danish Dairy Board (2004) has listed 37 mergers, acquisitions, takeovers, joint ventures and alliances in the global dairy industry. 72 Acta agriculturae slovenica, 84(december 2004)1. Turnover in billion US-Dollar Nestlé (Switzerland) .............................. Dean Foods (usa) :::::::::::::::::i 7.1 Dairy Farmers of America (USA) .......II....... 16.4 Arla Foods (Denmark/Sweden) ,,,,,,,/,,, ,,,|6.1 Danone (France) I I I I I I I I I I I I l-l-l60 Fonterra (New Zealand) I-I-I-I-I-I-I-I-I-I-I-I-TÜ5.8 Parmalat (Italy) ¦¦¦.¦.¦.¦.¦.¦.¦.¦.¦¦¦.¦.¦.¦!5.8 Kraft Foods (USA) ¦¦¦.¦.¦.¦.¦.¦¦¦. T"^^] 5.3 Lactalis (France) ............ • ] 5.2 Unilever* (Netherlands/UK) ¦¦¦¦¦¦ ¦¦T^T14.9 Friesland Coberco (Netherlands) ......... ¦ ,| 4.3 Meiji Dairies (Japan) ......... ¦ 14.2 Bongrain (France) ¦------¦ 13.7 Morinaga (Japan) ¦------¦ 13.7 Campina (Netherlands) ........ 13.6 Land O´Lakes (USA) ......| 2.9 Sodiaal (France) . . . . . .| 2.6 Humana (Germany) . . . . . |2.4 Schreiber Foods* (USA) ¦.-.¦.¦.¦.¦!2.4 Nordmilch (Germany) -.-.-.-.- .| 2.2 Source: Own presentation based on Rabobank International, 2003 Figure 5. The Top-20 dairy processing companies in 2002/2003. Slika 5. Vodilnih 20 mlečno predelovalnih podjetij v letih 2002/2003. The driving forces of the concentration process/potential advantages of large enterprises and/or plants In the management literature many driving forces, but also some limitations of the concentration process are mentioned and discussed (Schmidt, 1995, Weindlmaier 1999 and 2001). A first important advantage of large enterprises is the chance to realise economies of scale, respectively lower average costs. These cost reductions can have several origins. A first reason is technical economies as for example large dairy companies can use expensive modern technology and equipment. Secondly, managerial economies can occur in the administration of a large firm by splitting up management jobs and employing specialist accountants, salesmen, IT specialists, etc. Thirdly, financial economies frequently are realised by borrowing money at lower rates of interest than smaller firms. Fourthly, marketing economies are very important nowadays. These are achieved by spreading the high costs of advertising on television and in national newspapers across a large level of output. In Western Europe, today for a national TV-campaign at least 10 million Euros are required if a significant effect and growing sales can be expected. Commercial economies can be made when buying supplies in bulk and therefore gaining a larger discount. As an example, price deductions when buying large quantities of packing material, processed fruits or bacteria cultures can be named. Last but not least, research and development economies are important when developing new and better products. Investigations show that only large enterprises are able to invest in technical equipment and to employ the necessary specialists for an efficient R&D. Furthermore, the high cost of introducing product innovations to the market (e.g. listing fees) presupposes a good financial basis of the company. Fig. 6 shows as an example the long run cost curve for the production of UHT-milk (excluding raw-material costs). If only 10 million packages of UHT-milk are produced per year, costs of more than 20 Cent per package accrue. In case of a yearly production of more than 180 million packages, the relevant costs are only 14.5 Cent per package, equal to a cost reduction of Weindlmaier, H. The consequences of changing conditions of the European dairy sector … dairy companies. 73 nearly 30 per cent. Of course, such a cost difference will be decisive for the competitiveness of a producer of UHT-milk. Cent per package 1 l UHT-milk 30.0 Total cost 28.0 26.0 24.0 22.0 20.0 18.0 16.0 14.0 12.0 10.0 0 20,000 40,000 60,000 80,000 100,000 120,000 140,000 160,000 180,000 200,000 , ,., iJC ,_ , . ^ , ^___„ 1000 packages Source: Institut für Ernährungswirtschaft, 2003._____________________________________________ Figure 6. Average long run costs of the production of UHT-milk (except raw milk). Slika 6. Povprečni stroški proizvodnje UHT mleka (razen surovega mleka). In the 1960's, management consultants at The Boston Consulting Group observed a consistent relationship between the costs of production and the cumulative production quantity, which is called the effect of Experience Curves (Wikipeda, 2004). Data revealed that the value-added costs decline by 15 to 25 percent each time cumulative volume doubles. The Experience Curve suggests a possibility that systematic cost differences might arise between competitors because increased activity leads to increased learning, which leads to lower costs. The experience curve has important strategic implications. If an enterprise is able to gain market share over its competitors, it can develop a cost advantage. Penetration pricing strategies and a significant investment in advertising, sales personnel, production capacity, etc. can be justified to increase market share and gain a competitive advantage. A further important driver of concentration is the advantage of large companies by realising long run strategies (Maucher, 1993). The point made is that only large enterprises have the opportunity to compensate the risks of different operations. An example is internationalisation of dairy companies, which frequently leads for several years to losses in the newly integrated markets. Only large companies are able to compensate these losses by the profits made in other countries. Large, diversified dairy companies can more easily adjust their production program to the actual market situation and the development of actual profit margins. That this flexibility can be advantageous could be observed in 1998/99: The sharp tumble of the prices for butter and standard cheese in the EU mainly affected those specialised dairies which had no production alternatives. A further very important driver for concentration of dairy companies is the high concentration and internationalisation of the food retail trade, which has been discussed before. Only large 74 Acta agriculturae slovenica, 84(december 2004)1. dairy enterprises with a high production potential are able to deliver the large quantities of products required by these trade giants. Limitations of structural changes/potential disadvantages of large enterprises and/or plants Internal Diseconomies of Scale might be a consequence when the firm has become too large and inefficient. This might be due to the fact that management becomes out of touch with the shop floor and some machinery becomes over-staffed. Also, sometimes decisions are not taken quickly and there is too much form filling. Further more, lack of communication in a large firm is quite frequent, which means that management tasks sometimes get done twice. Finally, poor labour relations may develop in large companies. External Diseconomies of Scale might occur because of the increasing collection area for milk with the consequence of rising transport costs. In model calculations we performed recently we investigated to what extent collection cost of milk would increase if very large dairy processing plants (with up to 7 billion litres per plant) would be established. An important outcome is that in case of cost efficient milk collection (e.g. collection around the clock, collection per farm only every second day, collection based on route planning) this increase in collection costs is to a high percentage offset by the decrease in processing costs in larger processing plants (Weindlmaier and Huber, 2003). An important further limitation is psychological and emotional resistance of the full-time and honorary management (e.g. in dairy co-operatives) against the concentration process. It has its origin in the fact that one frequent and necessary consequence of a merger is the reduction of management positions. Therefore, the persons taking the decisions for merger quite often eliminate their own position in the enterprise. Closely connected with this argument is the fact that the potential advantages of concentration can be realised only if consequently major internal adjustments are performed. These include the shutdown of plants and possibly investments in new, modern processing facilities, the reduction of personnel leading to high expenses for compensation payments, etc. Generally, nowadays the financing of such processes becomes an increasing obstacle. Summarizing and evaluating these different driving forces and limitations, it seems that the advantages outperform the limitations. Therefore, further growth of dairy plants and enterprises is very likely. In the future, dairy plants of several billion litres per plant are a realistic vision. Porter’s Generic Competitive Strategies In his famous book “Competitive Strategy” Michael Porter of the Harvard Business School has argued that a firm’s strengths ultimately fall into one of two headings: cost advantage and differentiation (Porter, 1998a; QuickMBA, 2003). By applying these strengths in either a broad or narrow scope, three generic strategies result: cost leadership, differentiation and the focus strategy. These strategies present an ideal vehicle to discuss the options dairy companies have to stabilise respectively increase competitiveness. Cost leadership strategy The cost leadership strategy focuses for being the low cost producer for a given level of quality. There are several prerequisites to carry out such a strategy. A cost leader must focus on having a high market share in the respective market and improving process efficiencies by increasing the size of operations and by optimal outsourcing of non core activities. Furthermore, the access to materials and production factors at low costs is important: In the case of the dairy industry cost components of specific importance are low raw milk costs and low costs for packaging materials. A strict cost management and the avoidance of small, marginal customers Weindlmaier, H. The consequences of changing conditions of the European dairy sector … dairy companies. 75 are also decisive. In addition, cost leaders tend to minimise the costs in the areas R&D, service, promotion, etc. If we take into account the changed conditions of the European dairy industry, the importance of the strategy of cost leadership increased remarkably in recent years. It is expected that in the future the production of standard dairy products, of private labels and products for the discount stores will be a domain of large companies able to perform cost leadership for the respective product group. In a recent strategy plan for the Irish Dairy Processing Sector for instance one of the main proposals has been to reduce the number of plants for the production of butter, powder and casein from eleven to four plants to improve cost efficiency (Prospectus and promar International, 2003). Because of increasing competition even producers of value added products, of brands and specialities nowadays must focus on cost management. The developments on the markets emphasize that in the future the expectable price premium for brands and specialities will allow rather small extra costs only. Differentiation strategy A differentiation strategy calls for the development of products that offer unique attributes. Customers perceive that the products offer features that are better than or different from the products of the competitors. The value added by the uniqueness of the products may allow the company to charge a premium price for it. An economic prerequisite is that the price differences between conventional and premium products exceed the relevant cost differences. Health Convenient use (drinking yoghurt, intermediate meals, snacking) Convenience Functional food/ wellness products/anti aging (probiotic products, biological products, additives of minerals and vitamins, low fat products) New diversity (exclusive desserts, food providing spoiling experiences) Target group oriented contents (kids, seniors, etc.) Pleasure Source: Own presentation based on Dölle and Weindlmaier, 2003. Figure 7. Actual areas for product innovations in the dairy sector. Slika 7. Aktualna področja inovacij proizvodov v mlekarstvu. Looking at dairy products, there are several possibilities to offer additional benefits. Important examples are the differentiation of the basic characteristics of the products itself, for instance by use of specific recipes or ripening cultures, by utilising specific raw materials and ingredients or 76 Acta agriculturae slovenica, 84(december 2004)1. by employing special processing techniques, allowing a longer shelf life. Fig. 7 illustrates some areas for product innovation in the dairy sector. Actually, there are three main trends in consumer behaviour to be considered if new products are developed: Product innovations have to be healthy and wellness oriented, they are of high quality and incorporate the promise of pleasure and enjoyment by eating them, and last but not least, one of their features is convenience. However, table 5 demonstrates that consumer attitudes differ extremely between countries. According to a recent survey, convenience for instance is very important in UK, while in France and Italy this characteristic of innovations is less significant. The health aspect is a very important characteristic of new products in Italy, Spain and UK, but it is less essential in France and Germany. Table 5. Variations in consumer behaviour in different EU-countries Preglednica 5. Razlike v obnašanju potrošnikov v različnih državah ES 6 FOOD LOGICS people % % 12501 FRANCE 2452 20 GERMANY 3295 26 ITALY 2560 20 SPAIN 1696 14 U.K. 2497 20 HEALTH 44 39 40 48 47 46 QUALITY 17 20 11 22 18 14 CULTURE 26 36 24 28 26 18 CONVENIENCE 24 35 43 39 44 61 INDULGENCE 13 11 13 14 9 16 CONTINUITY 45 47 48 48 43 37 Index calculated European avera upon ge High over-representation Index 120 Significant over-representation 105 > Index < 120 Close to European average 90 > Index < 105 Significant under-representation Index < 90 * = Own presentation based on Hasson and Dofour, 2002 A further fundamental approach to reach uniqueness is branding. Brands deliver key signals to the consumers and simplify the buying process. They allow to equip the product with a “unique selling proposition” and to differentiate it from the many varieties of dairy products being more or less exchangeable. However, to develop such a USP nowadays, functional properties of the product have to be supplemented by emotional properties and extensive advertisement. A third important prerequisite of differentiation are widespread promotional activities. Besides classical advertisement by TV, radio, print and posters, “below-the-Line” promotion by sponsoring, event marketing and marketing via internet is of growing importance, also for dairy brands. The basic problem associated with a differentiation policy is the fact that product innovation, branding and promotion are very costly. The big players in the dairy sector like Nestlé and Danone have huge yearly budgets for R&D and promotion. For example, the average yearly Weindlmaier, H. The consequences of changing conditions of the European dairy sector … dairy companies. 77 budget for R&D of Nestlé during the years 2000–2002 has been more than 770 million Euros. Furthermore, because of the described developments in the food retail trade and its introduction of own labels, the conditions for a branding policy by the dairy industry have deteriorated. Consequently, a differentiation strategy is for sure a very important option for the large, concentrated enterprises in the dairy sector. The risks associated with it include the growing speed of imitation by competitors and the fast changes in customer tastes leading to short life cycles of the products. For the many small and medium sized companies a differentiation strategy offers only limited or no perspectives. For this group of enterprises, the next strategy discussed, the focus strategy, might achieve better results. Focus strategy The focus strategy concentrates on market niches and within those it attempts to achieve either a cost advantage or differentiation. A firm using a focus strategy often enjoys a high degree of customer loyalty, and this entrenched loyalty discourages other firms from competing directly. A focus strategy might be successful, if it succeeds to offer suitable products focusing on the special needs of the group served. Examples are organic dairy products and special regional cheese varieties. Such cheese varieties are particularly frequently offered in the Southern member countries of the EU, like in France, in Spain and in Italy. In addition, regional brands of dairy products, based on traditional production processes with a high portion of manual work or environmental advantages in the production region might form the basis for a focus strategy. Such regional brands benefit from the recently intensified preference for products from the home region, having the advantage of special freshness and short distribution distances. An example is the market for dairy products in Austria after the accession to the EU in 1995. The Austrian dairy sector has managed it to a large extent to convince the Austrian consumers that Austrian dairy products are better because of the sound environmental conditions under which Austrian milk production is carried out. Fig. 8 shows that dairy products made in Austria still have a high market share in their home country in spite of the fact that they are frequently sold at higher prices than imported products. This leads to an important requirement of a focus strategy: Because of their narrow market focus, firms pursuing a focus strategy normally have lower volumes and therefore higher costs. Hence, they must be able to charge higher prices for their products. Taking into account the present market conditions and the increasing competition on the dairy markets, this becomes increasingly difficult. An example is the market for biological dairy products: Recently, we can observe a growing discrepancy between the high costs of production and distribution and the prices accepted by consumers. In spite of these drawbacks, for a limited number of SME’s a focus strategy might even in the long run guarantee the survival in competition to large enterprises. Still, this only will happen if the specific advantages forming the focus strategy can be preserved and if the price differences to conventional dairy products cover the additional cost (Burchardi and Thiele, 2003). Internationalisation There is a widespread view that an intensified internationalisation of the dairy industry is among the most important strategies to improve competitiveness. A first important argument in favour of this strategy is the growing internationalisation and globalisation of the markets for dairy products: Even companies, which in the past mainly sold their products regionally or nationally, are confronted with a growing number of competitors in their market area. Further important drivers are the preferences of European consumers, favouring a broad assortment of products from different countries and the already mentioned concentration and 78 Acta agriculturae slovenica, 84(december 2004)1. internationalisation of the European food retail trade. Last but not least the excess supply of dairy products in most of the European countries has to be considered. In spite of the European quota system it is a fact that the degree of self sufficiency for dairy products in the EU is about 108 per cent. Fig. 9 shows that in some of the European countries like Ireland, the Netherlands and Denmark high percentages of excess supply exist which is seeking for markets in other countries. Food retailers (except Hofer), Quantity until week 30/2003 97.8 96.6 99.3 86 6 88 81.8 f ** * S7> s> 4 L L <* if