Isolation and Identification of the Microbiota of Danish Farmhouse and Industrially Produced Surface-Ripened Cheeses

Klaus Gori,Mia Ryssel,Nils Arneborg,Lene Jespersen

doi:10.1007/s00248-012-0138-3

Abstract

For studying the microbiota of four Danish surface-ripened cheeses produced at three farmhouses and one industrial dairy, both a culture-dependent and culture-independent approach were used. After dereplication of the initial set of 433 isolates by (GTG)5-PCR 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 non-starter 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 surface-ripening culture. Moreover, Gram-negative 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 ripening.

Highlights

Cheeses harbour a complex microbiota characterised by a succession of different microorganisms during milk coagulation and ripening [24]
lactic acid bacteria (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]
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 Gram-positive coryneforms (Brevibacterium spp., Corynebacterium spp. and Microbacterium spp.)

Summary

Introduction

Cheeses harbour a complex microbiota characterised by a succession of different microorganisms during milk coagulation and ripening [24]. Lactic acid bacteria (LAB) starter cultures (e.g., mesophilic Lactocococcus lactis or thermophilic Streptococcus thermophilus) metabolise residual lactose and citrate to different aroma compounds [17]. Non-starter 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 [12]. 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 Gram-positive coryneforms (Brevibacterium spp., Corynebacterium spp. and Microbacterium spp.) [4]. Most recently one study has included the pyrosequencing technique for identification of cheese microbiota [38]

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