Isolation, characterisation and identification of lactobacilli focusing mainly on cheeses and other dairy products Valérie Coeuret, Ségolène Dubernet, Marion Bernardeau, Micheline Gueguen, Jean Vernoux To cite this version: Valérie Coeuret, Ségolène Dubernet, Marion Bernardeau, Micheline Gueguen, Jean Vernoux. Isolation, characterisation and identification of lactobacilli focusing mainly on cheeses and other dairy products. Le Lait, INRA Editions, 2003, 83 (4), pp.269-306. 10.1051/lait:2003019. hal-00895504 HAL Id: hal-00895504 https://hal.archives-ouvertes.fr/hal-00895504 Submitted on 1 Jan 2003 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Lait 83 (2003) 269–306 © INRA, EDP Sciences, 2003 269 DOI: 10.1051/lait:2003019 Review article Isolation, characterisation and identification of lactobacilli focusing mainly on cheeses and other dairy products Valérie COEURET, Ségolène DUBERNET, Marion BERNARDEAU, Micheline GUEGUEN, Jean Paul VERNOUX* Laboratoire de microbiologie alimentaire U.S.C INRA, Université de Caen Basse-Normandie, Esplanade de la Paix, 14032 CAEN Cedex, France Received 11 April 2002 – Accepted 5 May 2003 Published online 5 July 2003 Abstract – During the last fifteen years, the Lactobacillus genus has evolved and contains to date more than 80 species. They are present in raw milk and dairy products such as cheeses, yoghurts and fermented milks. Quality assurance programmes associated with research, development, production and validation of the health or technological benefits of these bacteria require their relevant isolation, counting and identification. This review presents the different selective media to isolate lactobacilli, and the numerous different available tools to characterise lactobacilli at genus, species or strain level using either culture-dependent methods: phenotypical, molecular or global methods, or using new culture-independent advanced molecular methods. Enzymes used for PFGE, hybridisation probes and PCR-based method primers are listed in seven tables. In conclusion, the main advantages and disadvantages associated with these techniques are presented. Lactobacillus / media / PFGE restriction enzyme / probe / primer / cheese / dairy product Résumé – Isolement, caractérisation et identification des lactobacilles des produits laitiers. Durant les quinze dernières années, le genre Lactobacillus a subi de nombreux remaniements et compte actuellement plus de 80 espèces. Les lactobacilles sont présents dans le lait cru, les produits laitiers tels que les fromages, les yaourts et les laits fermentés. Pour s’inscrire dans une démarche qualité visant au développement de lactobacilles à effet santé ou d’intérêt technologique, l’isolement, le comptage et la caractérisation parfaite de ces bactéries sont nécessaires. Cette analyse bibliographique présente les différents milieux sélectifs pour isoler les lactobacilles, ainsi que les outils disponibles à ce jour pour caractériser les lactobacilles au niveau du genre, de l’espèce ou de la souche, aussi bien par des méthodes culture dépendantes : phénotypique, moléculaire, globale, que par les nouvelles méthodes se réalisant à partir d’échantillons bruts. Les enzymes utilisées en PFGE, les sondes d’hybridation et les oligonucléotides utilisés pour les différentes techniques de PCR sont répertoriés en sept tableaux. En conclusion, les avantages et inconvénients de ces techniques sont présentés. Lactobacillus / milieu sélectif / enzyme PFGE / sonde moléculaire / amorce PCR / fromage / produit laitier * Correspondence and reprints E-mail: [email protected] 270 V. Coeuret et al. 1. INTRODUCTION: microflora of the dairy products (non- BIODIVERSITY OF LACTOBA- starter lactic acid bacteria: NSLAB) and CILLI IN DAIRY PRODUCTS originate from animals, farms and cheese dairies: Lb. casei ssp. casei/Lb. paracasei ssp. paracasei, Lb.rhamnosus, Lb. plantarum, Lactic acid bacteria (LAB) comprise a Lb. fermentum, Lb. brevis, Lb. buchneri, wide range of genera and include a consid- Lb. curvatus, Lb. acidophilus and Lb. pen- erable number of species. Their common tosus [43, 58, 83, 110, 120, 133]. NSLAB, traits are: Gram-positive, usually catalase- which are initially present in small num- negative, growth under microaerophilic to bers (102 to 103 NSLAB/g after pressing in strictly anaerobic conditions and lactic Cheddar cheese), increase to high numbers acid production. These bacteria are the in cheese varieties that require long ripen- major component of the starters used in ing times (107 to 108 bacteria/g within fermentation, especially for dairy prod- about 3 months in Cheddar cheese) [10, 21, ucts, and some of them are also natural 34, 58, 69, 73, 86, 110, 145]. components of the gastrointestinal micro- flora. Lactobacillus is one of the most Coppola et al. [44] studied the micro- important genera of LAB. In raw milk and biological characteristics of raw milk, nat- dairy products such as cheeses, yoghurts ural whey starter and cheese during the and fermented milks, lactobacilli are natu- first months of ripening of Parmigiano rally present or added intentionally, for Reggiano: thermophilic lactobacilli – Lb. technological reasons or to generate a helveticus, Lb. delbrueckii ssp. lactis. Lb. health benefit for the consumer. delbrueckii ssp. bulgaricus – disappeared within 30 d. Rod-shaped mesophilic facul- tatively heterofermentative lactobacilli – 1.1. Cheeses and milks Lb. casei, Lb. paracasei ssp. paracasei, Lb. paracasei ssp. tolerans and Lb. rhamnosus – In cheese, lactobacilli (Lb. helveticus and progressively increased in number until the Lb. delbrueckii ssp. and lactis) are present in fifth month of ripening. The results industrial starter cultures for processing showed that the thermophilic lactobacilli hard cheese (e.g. Emmental, Comté, Italian and Lb. rhamnosus were derived from nat- Grana and Argentinean hard cheeses) [21, ural whey starter, whereas the other com- 43, 92]. Pasta filata cheeses (Mozarella) ponents of non-starter lactobacilli were and Italian hard cheeses (Canestrato derived from raw milk. Similarly, Comté Pugliese and Parmigiano Reggiano) are cheese contains mesophilic lactobacilli also processed with Lb. delbrueckii ssp. strains. They originate from the raw milk, bulgaricus [42, 45, 83]. Lactobacilli start- and this source was probably more impor- ers are normally present at levels of tant than the factory environment [17, 58]. 109 bacteria/g, contribute to the lactic fer- Differences have been observed between raw mentation, and are involved at the beginning milk cheeses and pasteurised or microfiltered of ripening [70]. For example, in Emmen- milk cheeses [58, 70, 164]. Eliskases- tal cheeses, lactobacilli ferment galactose Lechner et al. [70] found that Bergkäse excreted by Streptococcus thermophilus, (Austrian regional cheese) cheeses made achieve acidification processes, and con- from pasteurised milk contained less than tribute to primary proteolysis [31, 35, 59, one-thousandth the number of facultatively 74]. Their numbers decrease rapidly during heterofermentative lactobacilli (FHL) present ripening, at a rate depending to some in raw milk cheeses. Differences in citrate degree on the sensitivity of the starters to metabolism, which occur in raw milk salt [110], on the water activity, and on the cheeses, can be attributed to the presence autolysis power of the strains [185]. Some of FHL in these cheeses. Very little effort is lactobacilli are also present in the natural currently devoted to controlling the numbers Identification of dairy lactobacilli 271 and types of NSLAB present, although Lb. paracasei and Lb. gasseri [88, 94, 113, these organisms are thought to have a sig- 142, 159]. For these products, careful nificant influence on cheese flavour devel- strain selection is necessary. Klein et al. opment and to participate directly in the [113] recently observed that the identity of production of some major aroma com- most of the lactobacilli used as probiotics pounds such as acetic acid, formic acid and differs from that marked on the packaging. gas [31, 56, 59, 173]. However, NSLAB Shah [162] reported that it is important to may also cause defects. For example, in monitor the survival of probiotic lactoba- Emmental cheeses, Lb. plantarum may cilli because a number of products have disturb the metabolism of propionic acid been found to contain only a few viable bacteria, resulting in lower quality cheeses bacteria by the time they reach the market due to opening and changes in flavour [87, 163, 167]. development [34]. Given the great potential economic It has repeatedly been claimed that the value of lactobacilli, one of the main objec- use of selected strains as adjunct cultures tives of microbiologists is to develop a improves and accelerates flavour develop- clear picture of the microflora present in Lactobacillus ment. The use of adjunct the various dairy products, and the way in cultures to Cheddar cheese or to cow’s which it changes during processing. For milk cheeses ripened for short periods of example, during cheese manufacture