Isolation and Identification of the Microbiota of

Authors: Klaus Gori Mia Ryssel Nils Arneborg Lene Jespersen

Publish Date: 2012/12/07

Volume: 65, Issue: 3, Pages: 602-615

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Abstract

For studying the microbiota of four Danish surfaceripened cheeses produced at three farmhouses and one industrial dairy both a culturedependent and cultureindependent approach were used After dereplication of the initial set of 433 isolates by GTG5PCR fingerprinting 217 bacterial and 25 yeast isolates were identified by sequencing of the 16S rRNA gene or the D1/D2 domain of the 26S rRNA gene respectively At the end of ripening the cheese core microbiota of the farmhouse cheeses consisted of the mesophilic lactic acid bacteria LAB starter cultures Lactococcus lactis subsp lactis and Leuconostoc mesenteorides as well as nonstarter LAB including different Lactobacillus spp The cheese from the industrial dairy was almost exclusively dominated by Lb paracasei The surface bacterial microbiota of all four cheeses were dominated by Corynebacterium spp and/or Brachybacterium spp Brevibacterium spp was found to be subdominant compared to other bacteria on the farmhouse cheeses and no Brevibacterium spp was found on the cheese from the industrial dairy even though B linens was used as surfaceripening culture Moreover Gramnegative bacteria identified as Alcalignes faecalis and Proteus vulgaris were found on one of the farmhouse cheeses The surface yeast microbiota consisted primarily of one dominating species for each cheese For the farmhouse cheeses the dominant yeast species were Yarrowia lipolytica Geotrichum spp and Debaryomyces hansenii respectively and for the cheese from the industrial dairy D hansenii was the dominant yeast species Additionally denaturing gradient gel electrophoresis DGGE analysis revealed that Streptococcus thermophilus was present in the farmhouse raw milk cheese analysed in this study Furthermore DGGE bands corresponding to Vagococcus carniphilus Psychrobacter spp and Lb curvatus on the cheese surfaces indicated that these bacterial species may play a role in cheese ripeningCheeses harbour a complex microbiota characterised by a succession of different microorganisms during milk coagulation and ripening 24 During cheese ripening lactic acid bacteria LAB starter cultures eg mesophilic Lactocococcus lactis or thermophilic Streptococcus thermophilus metabolise residual lactose and citrate to different aroma compounds 17 Later LAB starter numbers decrease with cell death and their subsequent lysis results in release of intracellular peptidases involved in proteolysis of peptides to free amino acids 8 31 54 Several free amino acids are flavour compounds themselves but more important free amino acids are precursors of other cheese flavour compounds including ammonia carbonyl and sulphur compounds Nonstarter LAB homo and heterofermentative lactobacilli and pediococci either present as indigenous milk microorganisms contaminants or added as ripening cultures will grow during ripening and in most cases enhance flavour intensity 12Surfaceripened cheeses are characterised by an additional ripening from the cheese surface to the interior due to the activity of both yeasts and bacteria on the cheese surface 7 During the initial ripening period yeasts primarily Debaryomyces hansenii for semisoft cheeses and additionally Geotrichum candidum for soft cheeses and coagulasenegative staphylococci Staphylococcus equorum are present 3 21 26 Generally D hansenii and staphylococci on cheese surface are assumed to originate from the cheese brine which often is not changed or pasteurised between salting of different batches 2 3 42 For D hansenii Petersen et al 46 showed that the dominating D hansenii strain on cheeses of the Danish Danbo type did not originate from the added ripening culture but from the dairy housemicrobiota present in the ripening room D hansenii is important during cheese ripening as it assimilates lactate and produces alkaline metabolites such as ammonia thereby increasing pH of the cheese surface 23 46 which enables the growth of the less acid tolerant bacterial microbiota primarily Grampositive coryneforms Brevibacterium spp Corynebacterium spp and Microbacterium spp 4 In addition subpopulations of bacteria such as Grampositive Marinilactibacillus spp and Gramnegative Halomonas spp Vibrio spp and Proteus spp and bacteria of the Enterobacteriaceae family have been reported to occur on cheese surfaces 14 15 25 34 35 41 50 The presence of Gramnegative bacteria was first hypothesised to be indicative of hygienic problems However more recent results have shown that they produce important cheese flavour compounds and thus might contribute positively to the cheese ripening process 11 Several studies have characterised the cheese microbiota by both culturedependent methods and independent methods 43 Following culture isolation identification has been based on either macro and micromorphological examinations and by genotypic identification based on DNA restriction amplification and sequencing Furthermore many culture independent methods eg denaturing gradient gel electrophoresis DGGE have been used to study microbial diversity in cheeses 27 Most recently one study has included the pyrosequencing technique for identification of cheese microbiota 38During the last decade increasing interest in highquality cheeses produced at Danish farmhouses has resulted in an increasing number of Danish farmhouses producing a large variety of cheeses of which many are surfaceripened Contrary to the microbiota of in particular German and French surfaceripened cheese varieties the microbiota of Danish surfaceripened cheeses have only been investigated to a limited extent 37 38 46The aim of the present study was to investigate the microbiota of three surfaceripened cheeses produced at three individual Danish farmhouses and one surfaceripened cheese produced at one Danish industrial dairy Investigations of the cheese microbiota composition are highly relevant as the cheese microbiota together with rennet and indigenous milk enzymes plays an important role for both the flavour aroma and taste and textural properties of the final product To our knowledge this is the first study which simultaneously identified the cheese microbiota including both bacteria and yeasts in samples taken separately from the cheese interior and surface by both culturedependent and independent approachesThe present study was conducted on three surfaceripened cheeses produced at three individual Danish farmhouses dairies A B and C and one surfaceripened cheese produced at one Danish industrial dairy dairy D Cheeses from dairy A and C were of the Havarti type whereas cheeses from dairy B and D were of the Danbo type All cheeses were made with mesophilic LAB starters and except for the cheese from dairy A made of pasteurized milk After brining cheeses from dairies A C and D were smeared with Brevibacterium linens whereas no commercial ripening cultures were used for the cheese from Dairy BFor the cheeses from dairies A and C sampling took place at the end of ripening which for both cheeses was 12 weeks whereas for the cheeses from dairy B and D sampling took place prior to the washing and paraffin treatment which was after 6 weeks of ripening One sample from each cheese was analysed Ten grams of cheese from the surface depth ~ 4 mm and the interior respectively was removed using a sterile scalpel and 2  w/v trisodium citrate was added to yield a 110 dilution in stomacher bag The mixture was homogenized using a Stomacher for 2 min at medium speed From this dilution 10fold dilutions were prepared in 09  w/v NaCl The interior bacterial microbiota was enumerated on M17 with 10  glucose GM17 incubated for 3–4 days aerobically at 30 °C and 37 °C respectively and on MRS pH 62 and 54 incubated for 3–4 days anaerobically at 30 °C and 37 °C respectively The surface bacterial microbiota was enumerated on tryptic soya agar TSA with 00  and 40  w/v NaCl respectively incubated for 10–12 days aerobically at 30 °C All media for bacterial enumeration were added 02  w/v sorbic acid Merck and 01  w/v cycloheximide Merck to suppress growth of moulds and yeasts The interior and surface yeast microbiota was enumerated on Malt Yeast Glucose Peptone MYGP agar composed of 30 g of malt extract Difco 30 g yeast extract Difco 10 g of glucose Merck 50 g Bactopeptone Difco and 15 g of agar Difco per litre of distilled water pH 56 incubated for 5–8 days aerobically at 25 °C MYGP was added 100 mg/l chloramphenicol and 50 mg/ml chlortetracycline Sigma St Louis MO USA to suppress bacterial growth Twenty to forty bacterial and yeast colonies were selected from countable plates and were purified by restreaking twice on the appropriate media For longterm storage purified isolates were stored at −80°C in appropriate media containing 20  w/v glycerolMoisture and salt contents were determined by standard methods 32 51 Water activities a w of grated cheese samples were measured using a Aqualab CX2 Decagon Devices USA Measurements of pH were performed by placing a surface electrode Inlab 426 MettlerToledo Glostrup Denmark connected to a pH meter 1120 MettlerToledo directly on the cheese samples Calibration of the electrode was performed in buffers with pH 401 and 700 Radiometer Brønshøj Denmark

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