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Cheese is the quintessential convenience food, which can be consumed as it is, without preparation, can be used as a sandwich filler, grated or diced and used as a condiment or as a component of several cooked dishes. An estimated 40% of cheese is used as an ingredient or component of other foods.

This article addresses general concepts surrounding the use of starter cultures in cheesemaking. Starter cultures are essential to the manufacture of many cheese types. Whether prepared from the natural flora of milk, propagated as cultured whey, or selected from a preserved collection of defined single strains or undefined mixtures, starter cultures must be matched to the cheese type being made. Cheese starter cultures are predominantly composed of lactic acid bacteria, although other bacteria and yeasts may also be involved. In cheese manufacture, the primary role of starter cultures is the production of lactic acid from lactose at a predictable and controlled rate. Controlling acid production during cheesemaking is key to achieving control over curd pH, moisture, and lactose level. These factors in turn have a major influence on the microbial, chemical, and biochemical environment of the maturing cheese. Metabolism by starter cultures and the enzymes they produce contribute to the development of cheese flavor. The preparation and use of bulk starter cultures and culture concentrates are discussed.

The coagulation of milk (by proteolysis or acidification) is the key operation in cheesemaking. The enzymatic (rennet-induced) coagulation of milk can be divided into two phases: (1) hydrolysis of the micelle-stabilizing protein, κ-casein, (2) aggregation and gelation of the rennet-altered micelles, with the development of a particulate gel.

The rheology of cheese characterizes its deformation behaviour when subjected to stress or strain. Based on stress/strain behaviour, materials may be generally classified as ideal elastic solids, ideal viscous (Newtonian) liquids, or viscoelastic. Cheeses, like most other solid- and semi-solid foods that contain moisture and solids such as protein, fat and/or carbohydrate, exhibit the characteristics of both an elastic solid and a viscous fluid, and are thus termed viscoelastic. The rheological behaviour of cheese can be measured by an array of tests. Some tests, for example creep and low strain oscillation rheometry, involve application of a low strain (e.g.

The objective of this chapter is to present a very brief description of the principal operations so that the operations described in the following chapters can be seen in an overall context.

This chapter considers the microbiology of cheese ripening and complements the next chapter which considers the biochemistry of cheese ripening and the development of cheese flavour. The important parameters controlling the shelf-life of cheese, viz., water activity, NaCl level, oxidation-reduction level, pH, nitrate and temperature are examined in some detail as is the growth of non-starter lactic acid bacteria, mainly lactobacilli, which grow in all cheeses during ripening. The role of the secondary cultures, e.g., brevibacteria, propionibacteria and moulds, which grow only during ripening, are considered within descriptions of the microbiology of the individual cheese varieties. The cheeses examined in detail from a microbiological view include Cheddar, Swiss-type cheese, Parmigiano Reggiano, Gouda and Edam, bacterial-, e.g., Limburger, Livarot and Tilsit, Reblochon and Gubbeen, and mould surface-ripened cheeses, e.g., Camembert and Brie, and blue cheeses. Microbial spoilage of cheese, e.g., early and late gas formation, open texture, growth of lactobacilli and propionibacteria in Dutch-type cheese, and yeast and moulds, is considered. Finally descriptions of the various genera other than starter and non-starter bacteria found in cheese, e.g., Agrococcus, Arthrobacter, Brachybacterium, Brevibacterium, Corynebacterium, Microbacterium, Propionibacterium, Micrococcus, Kokuria, Kytococcus, Staphylococcus and the various yeasts and moulds are given.

The salt content in rennet-curd cheeses ranges from ~0.7 % (w/w) in Swiss to ~5 % (w/w) in Domiati. Salt has three major functions in cheese: it acts as a preservative; it enhances safety and contributes directly to salty flavour. Together with the desired pH, water activity and redox potential, salt assists preservation of cheese by minimization of spoilage and preventing the growth of pathogens in cheese. The dietary intake of sodium in the modern western diet is generally excessive, being two to three times the level recommended for desirable physiological function (2.4 g Na, i.e., ~6 g NaCl per day). However, cheese generally makes a relatively small contribution to dietary Na intake except when large quantities of high salt cheeses, such as Domiati and Feta, are consumed.

The liquid remaining after removal of the fat and casein from milk by isoelectric or rennet-induced coagulation of the casein is called whey. The whey contains about 90 % of the water of milk, ~98 % of the lactose, ~25 % of the protein and ~50 % of the inorganic salts. Traditionally, whey was an essentially worthless by-product of the cheese industry, to be disposed of as cheaply as possible, e.g., as animal feed. However, lactose and the whey proteins have interesting and unique properties. Advances in protein isolation technology have made it possible to isolate and fractionate the whey proteins in undenatured form. Although of minor importance compared with sucrose, lactose has some important applications, especially in the production of infant formulae; in addition, it can be converted to a number of important derivatives. Human milk contains considerable quantities of unique oligosaccharides (OSs) which are believed to be significant for the development of the neonate. Bovine milk contains only low concentrations of OSs but these can be purified and concentrated from whey and there is considerable interest in developing commercially-viable processes.

Following rennet-induced gelation, the coagulum is subjected to a series of treatments (e.g., cutting, cooking, stirring, acidification, whey drainage), the principal objective of which is to encourage syneresis (removal of whey from the gel) and effectively to concentrate the casein and fat to the degree characteristic of the variety. The principal treatments, which are characteristic of the variety of cheese, are described in this chapter. The cheddaring and pasta filata steps, together with moulding, pressing and packaging cheese, are also discussed.

Fresh cheese represents a diverse group of varieties produced by the coagulation of milk, cream or whey via acidification, acidification with a small quantity of rennet, or a combination of acid and heat and which are ready for consumption once manufacturing is complete. The essential steps in the manufacture of acid-coagulated (or acid-curd) and acid/rennet coagulated verities involves slow quiescent acidification of the standardized, pasteurized cheese milk to pH values (4.6–4.8) close to the isoelectric point of casein, cutting or gently braking the gel, and concentration of the gel to a curd using various means including pouring the broken gel onto cheese cloth or perforated moulds, mechanical separation or membrane filtration. The resultant cheese is then packaged cold (

This chapter summarises foodborne illness due to cheese. Since 1980, there have been 53 outbreaks of food poisoning due to the consumption of cheese during which time almost 250,000,000 tonnes of cheese were produced. The most common organisms involved were Listeria monocytogenes, enteropathogenic Escherichia coli, particularly 0157:H7, Salmonella and Staphylococcus aureus. Of these, listeriosis is the most serious since many outbreaks have resulted in fatalities. The factors controlling the growth of pathogens are the same as those controlling the growth of starters and non-starter lactic acid bacteria in cheese (see Chap. 6). Generally, soft cheeses are more likely to be involved in cheese-associated outbreaks of foodborne illness than hard and semi-hard cheese. Generally, no details of the compositional data of the cheese involved in an outbreak are given, e.g., pH, salt and moisture; such information could be important in understanding how outbreaks occurred. Each of the four groups of bacteria involved is considered in some detail regarding their origin, the symptoms of the illness and the experimental evidence for their growth in cheese. Many cheeses are made from raw milk and confounding factors other than the use of raw milk were involved in these outbreaks.

Cheese quality relates to, and depends on, many attributes, including appearance, texture, functionality, flavour, safety and nutritional value, the relative importance of which depend on the application of the cheese. Since cheese is the end-product of a long and complex process, which may last for two or more years, its quality depends on many factors, some of which are generally pertinent but some are variety-specific. The most important are: 1. The milk supply, including its composition, microbial quality, preparation (standardization and heat treatment) and consistency. 2. The bacterial culture(s) used for acidification and which play(s) major roles during ripening. 3. The rennet used to coagulate the milk and which is the principal proteolytic agent during ripening. 4. The non-starter bacteria which are either indigenous to the milk or gain entry to the milk or cheese from the environment during manufacture. 5. Composition of the cheese. 6. Ripening of the cheese curd, including temperature and duration.

Cheese is a very diverse group of foods (perhaps as many as 1500 varieties). To help consumers, retailers and cheese technologists, several schemes for the classification of cheese have been proposed and used. Criteria for classification include: coagulating agent (rennet or acid); texture/moisture content (very hard, hard, semi-hard, semi-soft, soft); matured or fresh; microflora (internal bacterial, surface/smear bacterial, internal or surface mould, propionic acid bacteria).

The development of pasteurised processed (also called process) cheese products (PCPs) in the early 1900s was motivated by the need to develop cheese-like products that were stable (i.e., did not leak fat, ‘sweat’ or become greasy) at ambient temperatures (≤40 °C) and could be stored for a long time without a change in quality. Today, PCPs are used mainly as ingredient cheese products with customized functionalities, and to a lesser extent as table cheese products (e.g., processed cheese spreads and slices). They are produced by comminuting, melting and emulsifying, into a smooth homogeneous molten blend, one or more varieties of natural cheese and optional ingredients using heat, mechanical shear and (usually) emulsifying salts (ES). The optional ingredients permitted are determined by the type of PCP, as defined by national legislation. Manufacture involves formulation, size reduction of cheese and blending of ingredients, heating to 75–85 °C while continuously shearing until a hot uniform molten mass is obtained, hot-filling into packages and cooling. While the ES are not emulsifying agents per se, they solubilise the cheese protein which binds the free water and emulsifies the free fat released during processing (heating and shearing). The ES, usually sodium citrates or sodium phosphates, mediate protein solubilisation by upward adjustment of the pH and sequestering calcium from the cheese protein. PCPs are packaged in varying formats, for example retail products are available as foil-covered blocks or triangle portions, individually wrapped- or stacked-slices or tubs of spread, while products for the catering trade are available as sliceable blocks, slabs, sausage form or spreadable products filled into drums or buckets. The texture, cooking attributes and overall quality of PCPs are influenced by many parameters including characteristics of the cheese and optional ingredients used in formulation, processing conditions (heat, shear) and composition.

Milk is a very complex fluid. It contains four principal constituents, water, lipids, proteins and lactose and perhaps 100 minor constituents, the most important of which from a cheesemaking viewpoint is calcium phosphate. The manufacture and quality of cheese depend, especially, on the properties of one of its protein groups, the caseins, and to a lesser extent on the lipids. Most (~90 %) of the water of milk is removed in the whey, which contains the soluble constituents, i.e., the whey proteins, lactose and some of the inorganic salts. Traditionally, whey was an almost worthless by-product but it is now the source of several very valuable products which are described in Chap. 22.

While most natural cheeses are consumed directly as table cheese, which can be eaten directly or with crackers or bread, they are also used extensively as ingredients in culinary dishes. Nevertheless, cheeses are in many cases manufactured specifically for use as an ingredient rather than as table cheese. The manufacture of ingredient cheeses involves protocols which impart specific functionalities, such as controlled textural/rheological properties (e.g., sliceability, shreddability or crumbliness) and cooking properties (e.g., Mozzarella with customized flow and stringiness suited to specific pizza brands). Ingredient cheeses are used in an array of culinary dishes, formulated food products and ready-prepared meals. The types and level of functional attributes required from ingredient cheeses depend on the application in which they are used. The functionalities of the unheated and heated cheese are key quality determinants of ingredient cheese. These are strongly influenced by micro- and macrostructure. At a microstructural level, rennet-curd cheese is a matrix comprised of a calcium phosphate para-casein network, which imbibes the cheese serum (moisture and dissolved solids) and encases the fat phase. The network may be viewed as a polymer network, in which the casein polymers are cross-linked mainly by calcium and calcium phosphate. The degree of polymer cross-linking and the relative proportion of fat in the network control the response of the unheated cheese matrix to stresses and strains encountered during the size-reduction processes involved in shredding, grating or eating, and the response of the heated cheese during baking and grilling. At the macrostructural level, cheese is an assembly of fused curd particles (microstructures), with the extent of fusion depending on both the microstructure of the curd particles and the processes to which the curd particles are subjected such as salting, moulding, texturizing and pressing. Hence, a key approach in designing ingredient cheeses with target functionalities is the control of cheesemaking operations that affect the microstructure and macrostructure.

Agriculture dates from about 6000 bc, when certain plants and animals were domesticated in Mesopotamia. Among the domesticated animals were goats, sheep and cattle, the milk of which was consumed by Man as a high-quality nutrient. Milk is also a very good growth substrate for bacteria, some of which produce lactic acid, which causes the milk to gel. The acidified milk was consumed as cultured milk or converted to acid-curd cheese. It was also discovered that milk could be coagulated by certain proteolytic enzymes, e.g., chymosin from the stomach of neonatal mammals; the coagulum was converted to rennet-curd cheese. Cheese has been produced since the earliest civilizations, e.g., Sumer and Egypt and was well established in Classical Rome. Cheese production spread throughout Europe and the Middle East and later to North and South America and Oceania and evolved as at least 1000 varieties. Cheese production was a farm-based operation until the mid-nineteenth century, and much cheese is still produced at an artisanal level. However, the principal varieties are now produced in very large highly mechanized factories by highly developed technology.

Milk is a highly nutritious medium which can be easily contaminated by microorganisms. In this chapter, the sources of microorganisms, particularly bedding, faeces, the milking machine and milk storage tank are considered as is the role of disease, particularly mastitis. The most important source of contamination is the milking machine and bulk storage tank and the most important bacteria are psychrotrophs, i.e., bacteria that can grow relatively rapidly at

Enterococci and lactobacilli are ubiquitously found in the intestinal microflora of humans and animals. The aim of the present study was to determine the importance of bovine faeces as a source of these organisms in raw milk. One hundred and fifty six putative enterococci and 362 lactobacilli were isolated from bovine faeces (n=26), cows' teats, raw milk, the milking machine and the milking environment on one farm. The clonal relationships of each group were investigated using Pulsed-Field Gel Electrophoresis and representatives of the different clusters were identified by repetitive DNA element (rep)-PCR fingerprinting, protein profiling, phenylalanyl-tRNA synthase (pheS) sequence analysis or 16S rDNA gene sequencing. Lactobacilli were present at approximately 3 orders of magnitude greater than enterococci in the bovine faeces. The majority of the bovine faecal enterococcal isolates were identified as Aerococcus viridans. Seven teat isolates belonged to a potential novel Aerococcus sp. and one bovine faecal isolate to a potential second novel Aerococcus sp. The lactobacilli present in the bovine faeces were predominantly Lactobacillus mucosae and Lactobacillus brevis, with small numbers of Lactobacillus plantarum. Only one Enterococcus (a strain of E. casseliflavus) out of 76 and one Lactobacillus (a strain of L. parabuchneri/kefir) out of 247 of the bovine faecal isolates was found in the milk. The major source of these bacteria in the milk was the milking equipment.

Enterococcus faecalis FAIR E-239, growing on glucose plus citrate, metabolized citrate at pH 6.5 or 7.5, but only when glucose had been exhausted; it did not metabolize citrate at pH 5.5 or 8.5. When grown on citrate only, the strain metabolized citrate at all pH values, and two growth rates were apparent. Citrate was mainly metabolized during the second, much slower growth rate. Glucose also inhibited citrate metabolism by E. faecalis FAIR E-237 and FAIR E-259 and Enterococcus faecium FAIR E-338 and FAIR E-371. Glucose-grown resting cells were unable to metabolize citrate. Citrate-grown resting cells had a pH optimum of 4.7 for citrate metabolism but also metabolized significant amounts of citrate at pH 4.2 and 6.5. Resting stationary phase cells used citrate more rapidly than resting log phase cells. Citrate metabolism was faster at citrate levels

Enterococci are one of the major facultative anaerobic bacterial groups that reside in the human gastrointestinal tract. In the present study, the composition of the enterococcal fecal flora in three healthy humans was analyzed before, during, and after the daily consumption of ∼125 g of a raw-milk Cheddar-type cheese containing 3.2 × 10 4 enterococci/g of cheese. Enterococcal counts ranged between 1.4 × 10 2 and 2.5 × 10 8 CFU/g of feces and differed from subject to subject and from week to week. The cheese contained mainly Enterococcus casseliflavus and a small population of Enterococcus faecalis . Clonal relationships were determined by pulsed-field gel electrophoresis. Before and after consumption of the cheese, samples from humans contained mainly Enterococcus faecium , with some of the clones being resident. During consumption of the cheese, one particular transient clone of E. faecalis , clone Fs2, which was present in small numbers in the cheese, largely dominated the feces. Two clones of E. casseliflavus from the cheese were also found in the feces of one of the subjects during cheese consumption. These results suggest that a clone need not be present in a food in high numbers to establish itself in the intestine.

Growth of P. freudenreichii DPC 3801 in whey was stimulated by peptone, tryptone, pre-growth of Lactobacillus helveticusDPC 4571 in milk and by sodium caseinate hydrolysed with the crude proteinase of Lb. helveticusDPC 4571. Addition of vitamins (riboflavin, thiamine, PABA, Ca panthothenate, biotin and nicotinic acid) and minerals (MgSO4 and MnCl2 ) to control whey did not improve the growth of P. freudenreichii .C oCl 2 and CuSO4 inhibited growth. Growth of the lactobacillus in milk resulted in significant increases in peptide and amino acid production but the amino acids produced did not stimulate the growth of the PAB. Several chromatographic procedures, including ion-exchange, gel permeation and reverse-phase, high-pressure liquid chromatography failed to categorically identify the peptide(s) responsible for the stimulation of the PAB. In some of these chromatographic systems, the stimulatory activity was shown to be present in several peaks im- plying that different peptides were involved. Several tetra-, penta- and hexa-peptides produced by other strains of Lb. helveticus from αs- and β-casein had a small but significant effect on growth of P. freudenreichii DPC 3801. Based on these results it was concluded that stimulation of P. freudenreichii was due to production of peptides by Lb. helveticus from casein. Propionibacterium freudenreichii / Lactobacillus helveticus / peptide / whey

Comprehensive collaborative studies from our laboratories reveal the extensive biodiversity of the microflora of the surfaces of smear-ripened cheeses. Two thousand five hundred ninety-seven strains of bacteria and 2,446 strains of yeasts from the surface of the smear-ripened cheeses Limburger, Reblochon, Livarot, Tilsit, and Gubbeen, isolated at three or four times during ripening, were identified; 55 species of bacteria and 30 species of yeast were found. The microfloras of the five cheeses showed many similarities but also many differences and interbatch variation. Very few of the commercial smear microorganisms, deliberately inoculated onto the cheese surface, were reisolated and then mainly from the initial stages of ripening, implying that smear cheese production units must have an adventitious “house” flora. Limburger cheese had the simplest microflora, containing two yeasts, Debaryomyces hansenii and Geotrichum candidum , and two bacteria, Arthrobacter arilaitensis and Brevibacterium aurantiacum . The microflora of Livarot was the most complicated, comprising 10 yeasts and 38 bacteria, including many gram-negative organisms. Reblochon also had a very diverse microflora containing 8 yeasts and 13 bacteria (excluding gram-negative organisms which were not identified), while Gubbeen had 7 yeasts and 18 bacteria and Tilsit had 5 yeasts and 9 bacteria. D. hansenii was by far the dominant yeast, followed in order by G. candidum , Candida catenulata , and Kluyveromyces lactis . B. aurantiacum was the dominant bacterium and was found in every batch of the 5 cheeses. The next most common bacteria, in order, were Staphylococcus saprophyticus , A. arilaitensis , Corynebacterium casei , Corynebacterium variabile , and Microbacterium gubbeenense . S. saprophyticus was mainly found in Gubbeen, and A. arilaitensis was found in all cheeses but not in every batch. C. casei was found in most batches of Reblochon, Livarot, Tilsit, and Gubbeen. C. variabile was found in all batches of Gubbeen and Reblochon but in only one batch of Tilsit and in no batch of Limburger or Livarot. Other bacteria were isolated in low numbers from each of the cheeses, suggesting that each of the 5 cheeses has a unique microflora. In Gubbeen cheese, several different strains of the dominant bacteria were present, as determined by pulsed-field gel electrophoresis, and many of the less common bacteria were present as single clones. The culture-independent method, denaturing gradient gel electrophoresis, resulted in identification of several bacteria which were not found by the culture-dependent (isolation and rep -PCR identification) method. It was thus a useful complementary technique to identify other bacteria in the cheeses. The gross composition, the rate of increase in pH, and the indices of proteolysis were different in most of the cheeses.

Phenotypic and phylogenetic studies were performed on 11 strains of a Microbacterium-like organism isolated from the surface of a smear-ripened cheese. The isolates were Gram-positive, catalase-positive, facultatively anaerobic, oxidase-negative, non-spore-forming, non-motile, small, slender rods and grew in 12% (w/v) NaCl. Chemotaxonomic investigation revealed that all the isolates belonged unambiguously to the genus Microbacterium. They contained type B1 peptidoglycans with L-lysine as the diamino acid and glycolyl acyl types; rhamnose and galactose were the cell wall sugars. The G+C content ranged from 69 to 72 mol%. The major menaquinones were MK-11 and MK-12 and the major fatty acids were anteiso C15:0 and C17:0 and iso C16:0. Phylogenetic analysis of the 16S rRNA sequences of four isolates showed that they represented a new subline in the genus Microbacterium, with Microbacterium barkeri as their nearest phylogenetic neighbour. M. barkeri showed the highest sequence similarity to the isolates; however, DNA-DNA hybridization showed that the isolates had only 38% chromosomal similarity to M. barkeri. Based on the phylogenetic and phenotypic distinctiveness of the isolates, it is proposed that they be classified as a new Microbacterium species, for which the name Microbacterium gubbeenense sp. nov. is suggested. The type strain has been deposited as LMG S-19263T (= NCIMB 30129T). The GenBank accession number for the 16S rDNA sequence of the type strain is AF263563.

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).

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.

Growth of propionibacteria in complex media was independent of the initial number of cells; in contrast, growth of propionibacteria in milk and whey did not occur if the initial level of cells was 6 cfu/ml. Addition of vitamins, minerals or complex nitrogen sources to the milk or whey, or incubation under anaerobic conditions had no effect on the lack of growth. Addition of freeze-dried whey, prepared from skim milk reconstituted from powder, to a complex medium prevented growth from low inocula in the complex medium, demonstrating the presence of an inhibitor or inhibitors in the whey. The inhibitor(s) was heat stable, had a low molecular mass and retained its activity for at least 4 weeks at 20 °C. Pregrowth of some lactic acid bacteria, used as starter cultures in Swiss-type cheese manufacture, in milk for 2 weeks at 20 °C removed the inhibition, which explains how propionibacteria develop in Swiss-type cheese from low numbers even though they are inhibited in milk.

Haemin, Fe3+ and Cu2+ caused significant decarboxylation of 2-acetolactate (ALA) to diacetyl during steam distillation of milk at pH 6.5. In the absence of these compounds, maximum conversion (28%) of ALA to diacetyl occurred at pH 3.5. Addition of 1.5 m m CuSO4 at this pH increased the conversion to 100%. Based on this result, a new method for the determination of ALA was developed, in which interference from pre-formed acetoin was minimised. Comparison of this method with the previous method (Jordan and Cogan, Irish Journal of Agricultural and Food Research 34, 39–47, 1995), with which high concentrations of acetoin interfere, was excellent (R2=0.96). Breakdown of ALA to diacetyl in the absence of Cu at pH 0.5 and 6.5 were 0.8 and 2.5%, respectively, implying that samples for diacetyl analysis should be adjusted to pH 0.5 before distillation if ALA is also suspected to be present. In quarg (pH 4.5) held at 4°C, ALA was degraded at a rate of 6.9–16.9 μmol L-1 day-1 and from 32 to 50% of the degraded ALA was converted to diacetyl during extended storage.

Strains of a Gram-positive bacterium were isolated from the Irish smear-ripened cheese Gubbeen, and assigned to a new species, Corynebacterium mooreparkense, in 2001. During a further study on the same cheese, no additional isolates from this species could be found. Instead, multiple isolates of its nearest phylogenetic neighbour, Corynebacterium variabile, were found. A first screening with rep-PCR and SDS-PAGE pointed to a similarity between C. mooreparkense and C. variabile. Following this peculiar result, attempts were made to collect all type strains deposited at different culture collections and all strains described by Brennan et al. [Int J Syst Evol Microbiol (2001) 51, 843-852]. Subsequently, 16S rRNA gene sequencing and DNA-DNA hybridizations were performed. All C. mooreparkense strains had a 16S rRNA gene sequence similarity of at least 99.5 % with C. variabile and the DNA-DNA relatedness was 95 %. On the basis of these results, it is concluded that C. mooreparkense is a later heterotypic synonym of C. variabile.

Diacetyl formation was linear with time and with protein concentration when a cell-free extract of Leuconostoc lactis NCW1 was added to a buffer system containing pyruvate, thiamine pyrophosphate and MgS4 (final concentrations 60 mM, 0.11 mM and 0.22 mM, respectively). No diacetyl was detected in the absence of pyruvate or cell-free extract and no increase in diacetyl formation was detected on the addition of acetyl-CoA. When 2-acetolactate (1.6 mM) was the substrate, autodecarboxylation to diacetyl and acetoin occurred under aerobic and anaerobic conditions. When cell-free extract was added, decarboxylation of 2-acetolactate to acetoin and diacetyl increased 4–6-fold, under aerobic and anaerobic conditions. When the cell-free extract was boiled, diacetyl formation from 2-acetolactate was reduced to the level of autodecarboxylation. The results suggest that diacetyl is formed enzymatically in the presence and absence of oxygen, as well as spontaneously, from 2-acetolactate.

Standardized milks, heated at 72-100 °C to denature ~5-63% of the whey protein, were ultrafiltered to yield retentates with protein and fat levels of ~18.5 and 14%, respectively. Retentates were converted into semi-hard cheeses using specialized coagulation and gel-cutting equipment, with scalding and further syneresis being carried out in conventional cheese vats. High heat treatment of milk necessitated an increase in set temperature, a reduction in set pH and higher scalding temperatures in the cheese vat. Cheese from milk heated at 72 °C for 15 s had a mean composition of ~39.8% moisture, 28% protein, 45.1% fat-in-dry matter, 3.5% salt-in-moisture ( S M) and an ex-brine (1 day) pH of 5.27. Increasing levels of whey protein denaturation (WPD) resulted in cheeses having higher moisture, S M, and whey protein levels, lower ex-brine pH values and lower rates of pH increase during a 182-day ripening period. Cheeses with high levels of WPD also showed poorer curd fusion and lower yield (fracture) values during ripening. Higher levels of denatured whey protein in cheese were associated with a higher degree of primary proteolysis. However, the levels of small peptides ( lt 1000 Da) and amino acids were higher in the control cheese and generally decreased with increasing level of denatured whey protein in the cheese. The concentrations of most free fatty acids, especially butyric, were higher in control cheese.

13 C nuclear magnetic resonance ( 13 C-NMR) was used to investigate the metabolism of citrate plus glucose and pyruvate plus glucose by nongrowing cells of Lactococcus lactis subsp. lactis 19B under anaerobic conditions. The metabolism of citrate plus glucose during growth was also monitored directly by in vivo NMR. Although pyruvate is a common intermediate metabolite in the metabolic pathways of both citrate and glucose, the origin of the carbon atoms in the fermentation products was determined by using selectively labeled substrates, e.g., [2,4- 13 C]citrate, [3- 13 C]pyruvate, and [2- 13 C]glucose. The presence of an additional substrate caused a considerable stimulation in the rates of substrate utilization, and the pattern of end products was changed. Acetate plus acetoin and butanediol represented more than 80% (molar basis) of the end products of the metabolism of citrate (or pyruvate) alone, but when glucose was also added, 80% of the citrate (or pyruvate) was converted to lactate. This result can be explained by the activation of lactate dehydrogenase by fructose 1,6-bisphosphate, an intermediate in glucose metabolism. The effect of different concentrations of glucose on the metabolism of citrate by dilute cell suspensions was also probed by using analytical methods other than NMR. Pyruvate dehydrogenase (but not pyruvate formate-lyase) was active in the conversion of pyruvate to acetyl coenzyme A. α-Acetolactate was detected as an intermediate metabolite of citrate or pyruvate metabolism, and the labeling pattern of the end products agrees with the α-acetolactate pathway. It was demonstrated that the contribution of the acetyl coenzyme A pathway for the synthesis of diacetyl, should it exist, is lower than 10%. Evidence for the presence of internal carbon reserves in L. lactis is presented.

SummaryThe kinetics of lactate dehydrogenase fromLeuconostoc lactisNCW1 were studied. The pH optimum for the enzyme depended on the concentration of pyruvate used in the assay and the enzyme displayed an ordered mechanism with respect to substrate binding. TheKmfor pyruvate and NADH and theVmaxof the enzyme decreased 20–, 30– and 6-fold respectively as the pH decreased from 8·0 to 5·0. No activators were found and none of the intermediates of the phosphoketolase pathway tested inhibited the enzyme. ATP, ADP, GTP and NAD+were inhibitory. The intracellular volume (Volin) and intracellular pH (pHin) decreased as the extracellular pH (pHex) decreased. Co-metabolism of citrate and glucose affected the Volinbut did not affect the pHin, which decreased by 0·6 units per unit change in pHex; at pH 7·0, the pHinand pHexwere equal. The results suggest that pHinmay play a role in determining the production of diacetyl and acetoin at low pH byLeuconostoc.

SummaryMesophilic lactic starters and thermophilic lactobacilli but notStreptococcus salivariussubsp.thermophilusgrew on the sodium lactate agar (SLA) used for estimating the numbers of propionic acid bacteria (PAB) in cheese. The addition of cloxacillin (4 μg/ml) to SLA inhibited the starter bacteria but had no effect on the PAB. It was possible to count low numbers of PAB in the presence of high numbers of starter bacteria. A correlation coefficient of 0·9 was obtained between the level of propionic acid and the counts of PAB in cheese (n= 40). A disadvantage of the medium is that other bacteria found in cheese (mesophilic lactobacilli, enterococci,Clostridium tyrobutyricum) also grow on it; however, these bacteria are easily distinguishable from PAB on the basis of size, colour and absence of catalase.

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.

The growth characteristics of five bacteria, Brevibacterium aurantiacum 1-16-58, Corynebacterium casei DPC 5298 T , Corynebacterium variabile DPC 5310, Microbacterium gubbeenense DPC 5286 T , and Staphylococcus saprophyticus 4E61, all of which were isolated from the surface of smear cheese, were studied in complex and chemically defined media. All of the coryneforms, except M. gubbeenense , grew in 12% salt, while B. aurantiacum and S. saprophyticus grew in 15% salt. All five bacteria assimilated lactate in a semisynthetic medium, and none of the coryneform bacteria assimilated lactose. Glucose assimilation was poor, except by S. saprophyticus and C. casei . Five to seven amino acids were assimilated by the coryneforms and 12 by S. saprophyticus . Glutamate, phenylalanine, and proline were utilized by all five bacteria, whereas utilization of serine, threonine, aspartate, histidine, alanine, arginine, leucine, isoleucine, and glycine depended on the organism. Growth of C. casei restarted after addition of glutamate, proline, serine, and lactate at the end of the exponential phase, indicating that these amino acids and lactate can be used as energy sources. Pantothenic acid was essential for the growth of C. casei and M. gubbeenense . Omission of biotin reduced the growth of B. aurantiacum , C. casei , and M. gubbeenense . All of the bacteria contained lactate dehydrogenase activity (with both pyruvate and lactate as substrates) and glutamate pyruvate transaminase activity but not urease activity.

Seven Gram-positive, coryneform bacteria with virtually identical whole-organism protein patterns were isolated from the surface of smear-ripened cheeses. Representatives of these strains were the subject of a polyphasic study designed to establish their taxonomic status. The organisms formed a distinct branch in the Microbacteriaceae 16S rRNA gene tree and were most closely related to members of the genus Agrococcus, sharing sequence similarities of 95.4–98.7 %. The chemotaxonomic profiles of the strains were consistent with their classification in the genus Agrococcus. The combined genotypic and phenotypic data show that the isolates should be classified in the genus Agrococcus as representatives of a novel species. The name Agrococcus casei sp. nov. is proposed for this taxon. Isolate R-17892t2T (=DSM 18061T=LMG 22410T) is the type strain of Agrococcus casei sp. nov.

Cheese flavour is the result of complex biochemical transformations attributed to bacteria and yeasts grown on the curd of smear-ripened cheeses. Volatile sulphur compounds (VSCs) are responsible for the characteristic aromatic notes of several cheeses. In the present study, we have assessed the ability of Kluyveromyces lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae strains, which are frequently isolated from smear-ripened cheeses, to grow and deacidify a cheese medium and generate VSCs resulting from L-methionine degradation. The Kluyveromyces strains produced a wider variety and higher amounts of VSCs than the S. cerevisiae ones. We have shown that the pathway is likely to be proceeding differently in these two yeast genera. The VSCs are mainly generated through the degradation of 4-methylthio-oxobutyric acid in the Kluyveromyces strains, in contrast to the S. cerevisiae ones which have higher L-methionine demethiolating activity, resulting in a direct conversion of L-methionine to methanethiol. The deacidification activity which is of major importance in the early stages of cheese-ripening was also compared in S. cerevisiae and Kluyveromyces strains.

Kluyveromyces lactis is one of the cheese-ripening yeasts and is believed to contribute to the formation of volatile sulfur compounds (VSCs) through degradation of l -methionine. l -Methionine aminotransferase is potentially involved in the pathway that results in the production of methanethiol, a common precursor of VSCs. Even though this pathway has been studied previously, the genes involved have never been studied. In this study, on the basis of sequence homology, all the putative aminotransferase-encoding genes from K. lactis were cloned in an overproducing vector, pCXJ10, and their effects on the production of VSCs were analyzed. Two genes, Kl ARO8.1 and Kl ARO8.2 , were found to be responsible for l -methionine aminotransferase activity. Transformants carrying these genes cloned in the pCXJ10 vector produced threefold-larger amounts of VSCs than the transformant containing the plasmid without any insert or other related putative aminotransferases produced.

The growth of five bacteria isolated from red-smear cheeses, Brevibacterium aurantiacum, Corynebacterium casei, Corynebacterium variabile, Microbacterium gubbeenense and Staphylococcus saprophyticus in mixed cultures with Debaryomyces hansenii on aseptic model cheese curd at 10 and 14 °C was investigated. At both temperatures, C. casei and Micro. gubbeenense had a longer lag phase than C. variabile, Brevi. aurantiacum and Staph. saprophyticus. In all cultures, lactose was utilised first and was consumed more rapidly at 14 °C than at 10 °C, i.e., 6 d at 14 °C and 10 d at 10 °C. This utilisation coincided with the exponential growth of Deb. hansenii on the cheese surface. Lactate was also used as a carbon source and was totally consumed after 21 d at 14 °C and ~90% was consumed after 21 d at 10 °C regardless of the ripening culture. Small differences (Brevi. aurantiacum and after 21 d for the other bacteria. Regardless of the organisms tested, colour development and colour intensity were also greater at 14 °C than at 10 °C. This study has provided useful information on the growth and contribution to colour development of these bacteria on cheese.