Timothy M. Cogan

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.

Characterization of the lactic acid bacteria in artisanal dairy products

The European Union is thanked for partly financing this project under ECLAIR contract CT-91-0064.

Contribution of the indigenous microflora to the maturation of cheddar cheese

Abstract Cheddar cheeses were made from raw milk, pasteurised milk (72°C, 15 s) or milk produced from skim milk which had been microfiltered using an Alfa-Laval MFS-1 MF unit and mixed with pasteurised cream (72°C, 30 s). Microfiltration (MF) reduced the total bacterial count (TBC) by > 99% and MF cheesemilk had a lower TBC than pasteurised milk; counts of non-starter lactic acid bacteria (NSLAB) were

Biochemical properties of enterococci relevant to their technological performance

A total of 129 E. faecium, E. faecalis and E. durans strains of food, veterinary and human origin were screened for biochemical properties relevant to their technological performance. Strains exhibited low milk acidifying ability and low extracellular proteolytic activity, with food origin and E. faecalis strains being generally more active. Their peptidase activities were low and mainly specific against glycine-proline- and glutamate-4-nitroanilide, while only food origin and E. durans strains showed broader substrate specificity. In contrast, their lipolytic activities were relatively higher; food and veterinary origin and E. faecalis strains were the most lipolytic. The post-electrophoretic detection of esterase activities showed that the esterolytic system of enterococci was rather complex. All species showed strain-to-strain variation in their ability to metabolise citrate and pyruvate, with E. faecalis strains being generally more active. The main volatile compounds produced in milk were acetaldehyde, ethanol and acetoin; generally, E. faecalis strains produced the highest concentrations. None of the strains decarboxylated histidine, lysine and ornithine, but the majority produced tyramine from tyrosine, independently of origin and species. In respect of most biochemical properties considered in this study, E. faecalis strains were generally more active compared to E. faecium and E. durans. This was also the case for the isolates of food origin compared to those of veterinary and human origin. Results obtained allow the selection of enterococci strains to be used as adjunct starters in food fermentations. However, a final selection should take into account the potential virulence factors of enterococci.

Biodiversity of the Bacterial Flora on the Surface of a Smear Cheese

ABSTRACT The bacteria on the surface of a farmhouse smear-ripened cheese at four stages of ripening (4, 16, 23, and 37 days) from inoculated (i.e., deliberately inoculated with Brevibacterium linens BL2) and noninoculated (not deliberately inoculated with B. linens BL2) cheese were investigated. The results show that, contrary to accepted belief, B. linens is not a significant member of the surface flora of smear cheese and no microbial succession of species occurred during the ripening of the cheeses. Of 400 isolates made, 390 were lactate-utilizing coryneforms and 10 were coagulase-negative Staphylococcus spp. A detailed analysis of the coryneforms was undertaken using phenotypic analysis, molecular fingerprinting, chemotaxonomic techniques, and 16S rRNA gene sequencing. DNA banding profiles (ramdom amplified polymorphic DNA [RAPD]-PCR) of all the coryneform isolates showed large numbers of clusters. However, pulsed-field gel electrophoresis (PFGE) of the isolates from the cheeses showed that all isolates within a cluster and in many contiguous clusters were the same. The inoculated and noninoculated cheeses were dominated by single clones of novel species of Corynebacterium casei (50.2% of isolates), Corynebacterium mooreparkense (26% of isolates), and Microbacterium gubbeenense (12.8% of isolates). In addition, five of the isolates from the inoculated cheese were Corynebacterium flavescens. Thirty-seven strains were not identified but many had similar PFGE patterns, indicating that they were the same species. C. mooreparkense and C. casei grew at pH values below 4.9 in the presence of 8% NaCl, while M. gubbeenense did not grow below pH 5.8 in the presence of 5 to 10% NaCl. B. linens BL2 was not recovered from the inoculated cheese because it was inhibited by all the Staphylococcus isolates and many of the coryneforms. It was concluded that within a particular batch of cheese there was significant bacterial diversity in the microflora on the surface.

Surface Microflora of Four Smear-Ripened Cheeses

ABSTRACT The microbial composition of smear-ripened cheeses is not very clear. A total of 194 bacterial isolates and 187 yeast isolates from the surfaces of four Irish farmhouse smear-ripened cheeses were identified at the midpoint of ripening using pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR, and 16S rRNA gene sequencing for identifying and typing the bacteria and Fourier transform infrared spectroscopy and mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis for identifying and typing the yeast. The yeast microflora was very uniform, and Debaryomyces hansenii was the dominant species in the four cheeses. Yarrowia lipolytica was also isolated in low numbers from one cheese. The bacteria were highly diverse, and 14 different species, Corynebacterium casei, Corynebacterium variabile, Arthrobacter arilaitensis, Arthrobacter sp., Microbacterium gubbeenense, Agrococcus sp. nov., Brevibacterium linens, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus saprophyticus, Micrococcus luteus, Halomonas venusta, Vibrio sp., and Bacillus sp., were identified on the four cheeses. Each cheese had a more or less unique microflora with four to nine species on its surface. However, two bacteria, C. casei and A. arilaitensis, were found on each cheese. Diversity at the strain level was also observed, based on the different PFGE patterns and mtDNA RFLP profiles of the dominant bacterial and yeast species. None of the ripening cultures deliberately inoculated onto the surface were reisolated from the cheeses. This study confirms the importance of the adventitious, resident microflora in the ripening of smear cheeses.

Manufacture of Cheddar cheese with and without adjunct lactobacilli under controlled microbiological conditions

Abstract Cheddar cheeses were manufactured under controlled microbiological conditions to study the influence of selected strains of mesophilic lactobacilli on proteolysis and flavour development. In each of two trials, a control cheese (containing only a Lactococcus starter) and four experimental cheeses (containing the Lactococcus starter and adjunct lactobacilli) were manufactured. The Lactobacillus inocula were Lb. casei ssp. casei (4 strains), Lb. casei ssp. pseudoplantarum (4 strains), Lb. curvatus (4 strains) or Lb. plantarum (2 strains). In the experimental cheeses, counts of lactobacilli ranged from 10 4 to 10 5 cfu g −1 at milling and increased to ~5 × 10 7 cfu g −1 after 4 weeks. Control cheeses remained free of lactobacilli for up to 97 days and thereafter the counts did not exceed ~5 × 10 5 cfu g −1 . Addition of lactobacilli positively influenced flavour acceptability of the cheese after ripening for 6 months at 7 °C. Cheeses manufactured with Lb. plantarum or Lb. casei ssp. pseudoplantarum adjuncts received the best grades. PAGE and RP-HPLC indicated only relatively minor differences in proteolysis between the control and experimental cheeses; however, the experimental cheeses showed higher levels of free amino acids, as well as differences in profiles of individual free amino acids, when compared to the controls. The addition of low numbers of selected strains of Lactobacillus spp. to cheesemilk, while having only a relatively minor influence on proteolysis, positively influenced the quality of Cheddar cheese.

Commercial Ripening Starter Microorganisms Inoculated into Cheese Milk Do Not Successfully Establish Themselves in the Resident Microbial Ripening Consortia of a South German Red Smear Cheese

ABSTRACT Production of smear-ripened cheese critically depends on the surface growth of multispecies microbial consortia comprising bacteria and yeasts. These microorganisms often originate from the cheese-making facility and, over many years, have developed into rather stable, dairy-specific associations. While commercial smear starters are frequently used, it is unclear to what degree these are able to establish successfully within the resident microbial consortia. Thus, the fate of the smear starters of a German Limburger cheese subjected to the “old-young” smearing technique was investigated during ripening. The cheese milk was supplemented with a commercial smear starter culture containing Debaryomyces hansenii, Galactomyces geotrichum, Arthrobacter arilaitensis, and Brevibacterium aurantiacum. Additionally, the cheese surface was inoculated with an extremely stable in-house microbial consortium. A total of 1,114 yeast and 1,201 bacterial isolates were identified and differentiated by Fourier transform infrared spectroscopy. Furthermore, mitochondrial DNA restriction fragment length polymorphism, random amplified polymorphic DNA, repetitive PCR, and pulsed field gel electrophoresis analyses were used to type selected isolates below the species level. The D. hansenii starter strain was primarily found early in the ripening process. The G. geotrichum starter strain in particular established itself after relocation to a new ripening room. Otherwise, it occurred at low frequencies. The bacterial smear starters could not be reisolated from the cheese surface at all. It is concluded that none of the smear starter strains were able to compete significantly and in a stable fashion against the resident microbial consortia, a result which might have been linked to the method of application. This finding raises the issue of whether addition of starter microorganisms during production of this type of cheese is actually necessary.

Constitutive nature of the enzymes of citrate metabolism in Streptococcus lactis subsp. diacetylactis

Four enzymes of citrate metabolism (viz. citrate lyase, acetolactate synthase, diacetyl reductase and acetoin reductase) were constitutively present in cells of several strains of Streptococcus lactis subsp. diacetylactis . In strain DRC1, which was studied in detail, diacetyl reductase and acetoin reductase were partly repressed and acetolactate synthase partly induced by growth on citrate. The stage of growth also affected the formation of each enzyme. The buffer species affected the activity of acetolactate synthase, diacetyl reductase and acetoin reductase.

Corynebacterium mooreparkense sp. nov. and Corynebacterium casei sp. nov., isolated from the surface of a smear-ripened cheese

Ten isolates each of two different bacterial species isolated from the surface of a smear-ripened cheese were found to exhibit many characteristics of the genus Corynebacterium. The isolates were Gram-positive, catalase-positive, non-spore-forming rods that did not undergo a rod/coccus transformation when grown on complex media. Chemotaxonomic investigation revealed that the strains belonged unambiguously to the genus Corynebacterium. Their cell walls contained arabinose, galactose and short-chain mycolic acids (C22 to C36) and their peptidoglycan contained meso-diaminopimelic acid. The G+C content of the DNA was 51-60 mol%. MK-9 (H2) was the principal menaquinone. The 16S rDNA sequences of four isolates of each bacterium were determined and aligned with those of other members of the coryneform group. Phylogenetic analysis showed that the strains represented two new sublines within the genus Corynebacterium; Corynebacterium variabile and Corynebacterium ammoniagenes were their nearest known phylogenetic neighbours. Corynebacterium variabile and Corynebacterium ammoniagenes showed the highest levels of sequence homology with the isolates; however, DNA-DNA hydridization studies indicated that the Corynebacterium strains isolated from the cheese smear did not belong to either Corynebacterium variabile or Corynebacterium ammoniagenes (26 and 46% chromosomal similarity, respectively). On the basis of the phylogenetic and phenotypic distinctiveness of the unknown isolates, it is proposed that the bacteria be classified as two new Corynebacterium species, for which the names Corynebacterium mooreparkense sp. nov. and Corynebacterium casei sp. nov. are proposed. Type strains have been deposited in culture collections as Corynebacterium mooreparkense LMG S-19265T (= NCIMB 30131T) and Corynebacterium casei LMG S-19264T (= NCIMB 30130T).