Biodiversity and anti-listerial activity of surface microbial consortia from Limburger, Reblochon, Livarot, Tilsit and Gubbeen cheese

Posters from IDF symposium on cheese: ripening, characterisation and technology -Prague (Czech Republic), March 21-25 2004

Teagasc: The microbiology of an Irish smear-ripened cheese (PDF format, 60KB)
BfAM: Surface Microflora of Tilsit-Cheese (PDF format, 360KB)
Caen: Surface microflora of Livarot, a traditional smear ripened cheese (PDF format, 170KB)
INRA: New Approach to Rebuild Subecosystems of Livarot Cheese Having Desired Aromatic and Technological Properties (PDF format, 250KB)
TUN: Surface Microflora of Limburger Cheese (PDF format, 150KB)

Project description

This is a European Commission Shared Cost Project involving 8 laboratories from 6 EU countries and 4 smear cheese manufacturers from 3 EU countries. The laboratories include:

Teagasc, Ireland
Bundesanstalt für Alpenländische Milchwirtschaft, Austria
Universite de Caen basse-Normandie, France
INRA, France
University of Newcastle, United Kingdom
University of Gent, Belgium
Technical University of Munich, Germany
Institut Technique Français Fromages, France
and the cheese manufacturers include:
Syndicat des Fabriants de Pont l'Eveque et de Livarot, France
Syndicat Interprofessionel du Reblochon, France
Tirol Milch, Austria
Bauer KG, Germany

It is financed from the Quality of Life Programme within the 5th Framework. Its acronym is SCM (Smear Cheese Microflora) and its contract number is QLK1-CT-2001-02228. The total cost of the project is €2,290,595 of which the EU will contribute 74% (€1,694,680). The project started on September 1st 2001 and runs until August 31st 2004 (36 months).

Background
Objectives
Description of Work
Expected Benefits
Partners
Progress to date

Background

About 165,000 tonnes of smear ripened cheeses are produced annually in Europe, mostly in France, Germany and Austria. These cheeses are characterised by the development of a mixed microflora of yeast and bacteria on the surface of the cheese which gives it its characteristic, red, glistening (hence, the name "smear") colour, and by an increase in the pH of the surface layer during ripening. The latter is generally believed to be due to metabolism of lactate to CO₂ and H₂O by the yeast, which, causes the pH of the surface to increase from 4.8 to a level (~pH 6.0) at which the bacteria can grow. In turn, the bacteria are involved in transforming S containing amino acids, especially methionine, into compounds, such as methional, methanethiol and its oxidation products, which are important in determining flavour in these cheeses.

The increase in the pH of these cheeses during ripening makes the cheese prone to growth of pathogens, particularly Listeria monocytogenes, and smear cheeses have been incriminated in several outbreaks of listeriosis. A recent survey by one of the partners showed that 13 and 5% of raw and pasteurised milk cheeses respectively are contaminated with L. monocytogenes, with some of them containing >10⁵ L. monocytogenes /cm².

The microbial ecology of the smear is complex and poorly understood. There has also been no systematic study on whether there is a progression of different micro-organisms on the surface of the cheese during ripening. For a long time it was thought that Brevibacterium linens was the main bacterium involved and Geotrichum candidum and Debaryomyces hansenii the main yeasts. For this reason, smear cheeses are often intentionally inoculated with B. linens, G. candidum and/or D. hansenii after brining, by dipping, spraying or washing the cheeses in a saline solution of the organisms; more traditionally, especially in Germany, the young cheeses are inoculated with smear from old or ripenened cheeses. This procedure has an inherent disadvantage that any pathogens present on the old smear are also inoculated on to the young cheeses. Many other bacteria, including Arthrobacter, Brachybacterium, Corynebacterium, Micrococcus, and Staphylococcus species, and yeasts, including Candida, Pichia, Rhodotorula,Torulaspora, Trichosporon and Yarrowia species, have also been isolated from the surfaces of these cheeses. Many of these microorganisms, particularly the bacteria, are difficult to identify using classical phenotypic analyses. They also require molecular and/or chemotaxonomic analyses and so many of them may have been mis-identified.

Objectives

To identify and characterize the major yeast and bacteria present on the surface of 5 cheeses [Limburger (Germany), Reblochon (France), Livarot (France), Tilsit (Austria) and Gubbeen (Ireland)] at different stages of ripening with the ultimate aim of determining the diversity in the microorganisms that are present
Identify strains of yeast which inhibit Listeria monocytogenes and exploit them commercially to prevent the development of listeria on cheese.

Description of work

Task 1

Characterisation of the surface microflora

Yeasts and bacteria will be isolated from the surface of Livarot, Limburger, Reblochon, Tilsit and Gubbeen cheese at 3 different stages (early, middle and late) of ripening. Strains previously isolated from Tilsit and Gubbeen cheeese and from smears, which have been previously shown to be inhibitory to listeria, will also be added to the collection. This is expected to result in 1 500 yeasts and 1 500 bacteria many of which will be similar and which will need to be dereplicated to more manageable numbers. The bacteria will be dereplicated using Pyrolysis Mass Spectrometry (PyMS) and the yeasts by Fourier-Transform Infrared Spectroscopy (FTIR). Representative bacteria and yeasts will then be characterised using a polyphasic approach of classical phenotypic tests, and chemotaxonomic and molecular techniques. Whether other culturable or unculturable bacteria are present on the smear will be studied using Denaturing Gradient Gel Electrophoresis (DGGE). This will also allow us to determine the effectiveness of the isolation media.

Task 2

Determination of the population dynamics of bacteria and yeast during cheese ripening.

Primers and probes for the major bacterial taxa detected in the smear will be designed and used to study the population dynamics of freshly made (new) isolates from frozen samples of the same cheese from which isolates were originally made in Task 1. This will also allow us to answer several questions including, are the same organisms dominant in each cheese? are some organisms unique to each cheese? does a progression of organisms occur on each cheese during ripening? The Single Strand Conformation Polymorphism (SCCP) technique will be used to determine the type of interactions, which occur between the bacteria and the yeasts. All of this information will clarify the microbiology of this important group of cheeses.

Task 3

Identifying yeasts which have antilisterial activity and exploiting them commercially.

The ability of all yeast isolates to inhibit L. monocytogenes will be determined. Those strains, which show inhibitory activity, will then be checked for their ability to inhibit representatives of the major species of bacteria present in the smear. Those yeasts which inhibit L. monocytogenes and which do not inhibit the other organisms in the smear will be evaluated in model cheesemaking experiments to determine their ability to inhibit Listeria monocytogenes. The best isolates will then be evaluated by the industrial partners in the commercial manufacture of Reblochon, Limburger, Tilsit, Livarot and Gubbeen cheeses to determine their ability to inhibit adventitious listeria on the surface of the cheese and their effect on overall cheese quality.

Expected Benefits

At the end of the project we hope to have accomplished the following:

Identified all the yeasts and bacteria found in five smear-ripened cheeses at different stages during ripening
Quantified the contribution of the different bacteria and yeasts to cheese ripening
Determined if a progression of different microorganisms during ripening occurs
Identified yeasts which inhibit L. monocytogenes
Determined their ability to produce good quality commercial cheese
Identified the inhibitory compounds produced by the yeasts and

Finally, transferred the knowledge and expertise necessary to exploit the inhibitory yeast to other European manufacturers of soft cheeses.

Partners

Prof. Tim Cogan (Co-ordinator) Dairy Products Research Centre, Teagasc, Fermoy, Co. Cork. Ireland Phone: + 353 25 42227 Fax: + 353 25 42340 Email: tcogan@moorepark.teagasc.ie	Prof. Dr. Siegfried Scherer, Technical University of Munchen, Institute für Mikrobiologie, FML Weihenstephan, D-85354 FREISING, Germany. Phone: + 49 8161 71 3516 Fax: + 49 8161 71 4512 Email: Siegfried.Scherer@wzw.tum.de

Dr. Hans Sebastiani, Bundesanstalt für Alpenländische Milchwirtschaft, Rotholz 50a, A-62000 ROTHOLZ, Austria. Phone: + 43 5244 62262 28 Fax: + 42 5244 62262 29 Email: hans.sebastiani@rotholz.bmlf.gv.at	Dr. J. F. Chamba, Instituut Technique Français de Fromages (ITFF), 419, Route des Champs Laitiers, BP 30, 74801 La Roche sur Foron Cedex, France. Phone: + 33 4 50 03 33 03 Fax: + 33 4 50 25 82 26 Email: itff.jfchamba@wanadoo.fr

Dr. Micheline Guéguen, Universite de Caen basse-Normandie, Laboratoire de Microbiologie Alimentaire, Esplanade de la Paix, 14032 CAEN Cedex, France. Phone: + 33 2 31 56 56 21 Fax: + 32 2 31 56 61 79 Email: gueguen@ibba.unicaen.fr	M. Francois Michel, Syndicat des Fabriants de Pont l'Eveque et de Livarot (SFPL), 82, Rue de Berniéres, 1400 CAEN Cedex France. Phone: + 33 2 31 85 50 93 Fax: + 33 2 31 50 17 31 Email: unil@wanadoo.fr

Dr. Francoise Irlinger, INRA, Laboratoire de Génie et de Microbiologie des Procédés Alimentaires, INA P-G, CBAI, 78850 THIVERVAL-GRIGNON, France. Phone: + 33 1 30 81 54 91 Fax: + 33 1 30 81 55 97 Email: irlinger@grignon.inra.fr	M. Bruno Mathieu, Syndicat Interprofessionel du Reblochon (SIR), Maison du Reblochon, Rue Saint Blaise, BP 55, 74232 THONES Cedex France. Phone: + 33 4 50 32 74 74 Fax: + 33 4 50 32 11 10 Email: sir@reblochon.fr

The EC Scientific Officer is
Dr. Antonio De Giulio,
European Commission,
Research Directorate-General Health,
Food and Environment,
Rue de la Loi 200,
B-1049 Brussels.
Belgium
Phone: + 32 2 299 5886
Fax: + 32 2 296 4322
Email: Antoni.Di-Giulio@cec.eu.int
Further information can be obtained from the Co-ordinator
Prof. Tim Cogan
Dairy Products Research Centre,
Teagasc,
Fermoy,
Co. Cork.
Ireland
Phone: + 353 25 42227
Fax: + 353 25 42340
Email: tcogan@moorepark.teagasc.ie

Prof. Mike Goodfellow, Microbial Resources Centre, Dept. Agric. & Environ Sciences, University of Newcastle upon Tyne, NEWCASTLE UPON TYNE, UK. NE1 7RU. Phone: + 44 191 222 7706 Fax: + 44 191 222 5228 Email: m.goodfellow@ncl.ac.uk	H. Andreas Geisler, Tirol Milch reg. Gen. M.b.H., Lattellapatz 1, A-6300 WÖRGL, Austria. Phone: + 43 5332 7801 16 Fax: Email: a.geisler@tirolmilch.at

Prof. Dr. Ir. Jean Swings, BCCM/LMG Bacteria Collection, Laboratory of Microbiology, University Gent, K. L. Ledeganckstraat 35, B-9000 GENT, Belgium. Phone: + 32 9 264 5116 Fax: + 32 9 264 5092 Email: Jean.Swings@rug.ac.be	H. Rüdiger Beduhn, J. Bauer KG, Tegernau 1-10, D-83512 WASSERBURG/INN, Germany. Phone: + 49 8071 109 210 Fax: + 49 8071 109 214 Email: r.beduhn@bauer-milch.de