T.P. Beresford

Recent advances in cheese microbiology

Microorganisms are an essential component of all natural cheese varieties and play important roles during both cheese manufacture and ripening. They can be divided into two main groups; starters and secondary flora. The starter flora, Lactococcus lactis, Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii used either individually or in various combinations depending on the cheese variety, are responsible for acid development during cheese production. Starters may be either blends of defined strains or, as in the case of many cheeses manufactured by traditional methods, composed of undefined mixtures of strains which are either added at the beginning of manufacture or are naturally present in the cheese milk. During cheese ripening, the starter culture, along with the secondary flora promote a complex series of biochemical reactions which are vital for proper development of both flavour and texture. The secondary flora is composed of complex mixtures of bacteria, yeasts and moulds, and is generally specifically associated with particular cheese varieties. In many cheese varieties, the action of the secondary flora contributes significantly to the specific characteristics of that particular variety. The secondary flora may be added in the form of defined cultures, but in many situations is composed of adventitious microorganisms gaining access to the cheese either from ingredients or the environment. During cheese manufacture and ripening, complex interactions occur between individual components of the cheese flora. Environmental factors within the cheese also contribute to these interactions. Elucidation of such interactions would greatly add to our understanding of the cheese ripening process and would enable a more targeted approach to starter/adjunct selection for cheese quality improvement. In the past, research in this area was dependent on classical microbiological techniques, which are very time consuming, not suitable for handling large numbers of isolates and generally not suitable to studies at sub species levels. However, developments in this area have recently undergone a major revolution through the development of a range of molecular techniques, which enable rapid identification of individual isolates to species and strain level. Application of such techniques to the study of cheese microbiology should lead to major advances in understanding this complex microbial ecosystem and its impact on cheese ripening and quality in the coming years.

The complex microbiota of raw milk.

Here, we review what is known about the microorganisms present in raw milk, including milk from cows, sheep, goats and humans. Milk, due to its high nutritional content, can support a rich microbiota. These microorganisms enter milk from a variety of sources and, once in milk, can play a number of roles, such as facilitating dairy fermentations (e.g. Lactococcus, Lactobacillus, Streptococcus, Propionibacterium and fungal populations), causing spoilage (e.g. Pseudomonas, Clostridium, Bacillus and other spore-forming or thermoduric microorganisms), promoting health (e.g. lactobacilli and bifidobacteria) or causing disease (e.g. Listeria, Salmonella, Escherichia coli, Campylobacter and mycotoxin-producing fungi). There is also concern that the presence of antibiotic residues in milk leads to the development of resistance, particularly among pathogenic bacteria. Here, we comprehensively review these topics, while comparing the approaches, both culture-dependent and culture-independent, which can be taken to investigate the microbial composition of milk.

Genome Sequence of Lactobacillus helveticus, an Organism Distinguished by Selective Gene Loss and Insertion Sequence Element Expansion

Mobile genetic elements are major contributing factors to the generation of genetic diversity in prokaryotic organisms. For example, insertion sequence (IS) elements have been shown to specifically contribute to niche adaptation by promoting a variety of genetic rearrangements. The complete genome sequence of the cheese culture Lactobacillus helveticus DPC 4571 was determined and revealed significant conservation compared to three nondairy gut lactobacilli. Despite originating from significantly different environments, 65 to 75% of the genes were conserved between the commensal and dairy lactobacilli, which allowed key niche-specific gene sets to be described. However, the primary distinguishing feature was 213 IS elements in the DPC 4571 genome, 10 times more than for the other lactobacilli. Moreover, genome alignments revealed an unprecedented level of genome stability between these four Lactobacillus species, considering the number of IS elements in the L. helveticus genome. Comparative analysis also indicated that the IS elements were not the primary agents of niche adaptation for the L. helveticus genome. A clear bias toward the loss of genes reported to be important for gut colonization was observed for the cheese culture, but there was no clear evidence of IS-associated gene deletion and decay for the majority of genes lost. Furthermore, an extraordinary level of sequence diversity exists between copies of certain IS elements in the DPC 4571 genome, indicating they may represent an ancient component of the L. helveticus genome. These data suggest a special unobtrusive relationship between the DPC 4571 genome and its mobile DNA complement.

Molecular approaches to analysing the microbial composition of raw milk and raw milk cheese.

The availability and application of culture-independent tools that enable a detailed investigation of the microbiota and microbial biodiversity of food systems has had a major impact on food microbiology. This review focuses on the application of DNA-based technologies, such as denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient gel electrophoresis (TTGE), single stranded conformation polymorphisms (SSCP), the polymerase chain reaction (PCR) and others, to investigate the diversity, dynamics and identity of microbes in dairy products from raw milk. Here, we will highlight the benefits associated with culture-independent methods which include enhanced sensitivity, rapidity and the detection of microorganisms not previously associated with such products.

Use of autolytic starter systems to accelerate the ripening of Cheddar cheese

Abstract The rapid release of intracellular enzymes due to autolysis of lactic acid bacteria in the cheese matrix post-manufacture is thought to play a role in the acceleration of cheese ripening. To investigate this hypothesis Cheddar cheese was manufactured using three related starter systems which varied with respect to their autolytic properties. Starter system A contained a blend of two Lactococcus lactis strains (223 and 227) which had a low level of autolysis. System B was identical to A but included an adjunct of a highly autolytic strain of Lactobacillus helveticus (DPC4571). System C consisted only of strain DPC4571 as starter. The cheeses were evaluated during ripening for key ripening indices including autolysis of starter cells by release of intracellular marker enzyme lactate dehydrogenase (LDH), composition, proteolysis and flavour development by descriptive sensory analysis. Populations of Lb. helveticus DPC4571 decreased rapidly in cheeses B and C and were not detected by 8 weeks. The level of starter culture autolysis proceeded in the order C≫B>A. Levels of proteolysis were elevated in cheeses B and C relative to A. Principal component analysis of the sensory data separated the character of cheese A from that of cheeses B and C. Cheeses B and C developed a unique ‘balanced’ ‘strong’ flavour early in ripening with a ‘caramel’ and ‘musty’ odour and ‘sweet’ ‘astringent’ flavour compared to cheese A. Hierarchical cluster analysis grouped C at 2 months with B at 6 and 8 months reflecting accelerated flavour development. Proteolytic and sensory data support the hypothesis that autolysis accelerates the rate of cheese ripening.

A rapid PCR based method to distinguish between Lactococcus and Enterococcus.

Phenotypic characterisation of Lactococcus and Enterococcus species remains unreliable as strains of both genera have been isolated which do not conform to the traditional criteria for separation of these genera. A bank of 131 isolates was phenotypically characterised by three methods: (a) traditional broth tests, (b) API Rapid ID 32 Strep and (c) BBL Crystal ID kits. Differences in genus designation between commercial kits were evident for 12 strains (9%), while 7 strains (5%) remained unidentified by either kit. Published 16S rRNA sequences were aligned and used to design genus-specific primers which, when used in separate PCR reactions, were capable of distinguishing all type strains of Lactococcus and Enterococcus. These primers did not react with known species of Streptococcus, Pediococcus, Lactobacillus, Leuconostoc or Tetragenococcus. Isolates which could not be identified by phenotype were assigned to either genus on the basis of the gene primers.

Combination of hydrostatic pressure and lacticin 3147 causes increased killing of Staphylococcus and Listeria

The use of hydrostatic pressure and lacticin 3147 treatments were evaluated in milk and whey with a view to combining both treatments for improving the quality of minimally processed dairy foods. The system was evaluated using two foodborne pathogens: Staphylococcus aureus ATCC6538 and Listeria innocua DPC1770. Trials against Staph. aureus ATCC6538 were performed using concentrated lacticin 3147 prepared from culture supernatant. The results demonstrated a more than additive effect when both treatments were used in combination. For example, the combination of 250 MPa (2.2 log reduction) and lacticin 3147 (1 log reduction) resulted in more than 6 logs of kill. Similar results were obtained when a foodgrade powdered form of lacticin 3147 (developed from a spray dried fermentatation of reconstituted demineralized whey powder) was evaluated for the inactivation of L. innocua DPC1770. Furthermore, it was observed that treatment of lacticin 3147 preparations with pressures greater than 400 MPa yielded an increase in bacteriocin activity (equivalent to a doubling of activity). These results indicate that a combination of high pressure and lacticin 3147 may be suitable for improving the quality of minimally processed foods at lower hydrostatic pressure levels.

Spatial and temporal distribution of non-starter lactic acid bacteria in Cheddar cheese

Aims: The aim of this work was to investigate the spatial and temporal distribution of species and strains of non‐starter lactic acid bacteria (NSLAB) within Cheddar cheese.

Invited review: Lactobacillus helveticus—A thermophilic dairy starter related to gut bacteria

The strain Lactobacillus helveticus DPC4571 has emerged as a promising flavor adjunct culture for Cheddar cheese given that it is consistently associated with improved flavor. The availability of the complete genome sequence of Lb. helveticus DPC4571 has enabled the search for the presence or absence of specific genes on the genome, in particular those of technological interest. Indeed, this analysis has facilitated a greater understanding into the functioning of lactic acid bacteria as a whole. The biochemical pathways of Lb. helveticus responsible for producing flavor compounds during cheese ripening are poorly understood but now with the availability of a complete genomic sequence are ripe for exploitation. Bioinformatic analysis of the genome of Lb. helveticus DPC4571 has revealed a plethora of genes with industrial potential including those responsible for key metabolic functions that contribute to cheese flavor development such as proteolysis, lipolysis, and cell lysis. In addition, it has been demonstrated that Lb. helveticus has the potential to produce bioactive peptides such as angiotensin converting enzyme inhibitory activity in fermented dairy products, demonstrating the therapeutic value of this species. A most intriguing feature of the genome of Lb. helveticus DPC4571 is the remarkable similarity in gene content with many intestinal lactobacilli, although originating from considerably different environments. Bioinformatic analysis demonstrated that 65 to 75% of genes were conserved between the commensal and dairy lactobacilli, which allowed key niche-specific gene sets to be described. This review focuses on the isolation, characterization, and exploitation of the Lb. helveticus species with particular emphesis on taking into consideration recent genome sequence data for Lb. helveticus and other Lactobacillus species.

The microbial content of raw and pasteurized cow milk as determined by molecular approaches

The microbial composition of raw and pasteurized milk is assessed on a daily basis. However, many such tests are culture-dependent, and, thus, bacteria that are present at subdominant levels, or that cannot be easily grown in the laboratory, may be overlooked. To address this potential bias, we have used several culture-independent techniques, including flow cytometry, real-time quantitative PCR, and high-throughput DNA sequencing, to assess the microbial population of milk from a selection of commercial milk producers, pre- and postpasteurization. The combination of techniques employed reveals the presence of a previously unrecognized and diverse bacterial population in unpasteurized cow milk. Most notably, the use of high-throughput DNA sequencing resulted in several bacterial genera being identified in milk samples for the first time. These included Bacteroides, Faecalibacterium, Prevotella, and Catenibacterium. Our culture-independent analyses also indicate that the bacterial population of pasteurized milk is more diverse than previously appreciated, and that nonthermoduric bacteria within these populations are likely to be in a damaged, nonculturable form. It is thus apparent that the application of state-of-the-art approaches can provide a detailed insight into the bacterial composition of milk and could potentially be employed in the future to investigate the factors that influence the composition of these populations.