"Some strains are positive. Source: Cogan, 1996.
Lactobacilli are the most acid tolerant of the lactic acid bacteria, preferring to initiate growth at acidic pH (5.5-6.2) and lowering the pH of milk to below 4.0. Lactobacilli are slow to grow in milk in pure culture. For this reason, they are generally used in combination with S. thermophilus.
Propionibacterium spp. are non-spore-forming, pleomorphic, gram-positive rods that produce large amounts of propionic and acetic acid and carbon dioxide from sugars and lactic acid. They are anaerobic to aerotolerant mesophils. They are not considered to belong to the lactic acid bacteria, but are closely related to coryneform bacteria in the Actinomycetaceae group. Four species of Propionibacterium are found in cheese (Table 3), but P. freudenreichii subsp. freudenrei-chii and P. freudenreichii subsp. shermanii are most often used in cheese manufacture (Lyon and Glatz, 1995). Although Propionibacterium spp. are found in raw milk, they may be present in insufficient numbers to produce an adequate fermentation, so they are often added along with the lactic culture when cheese with eyes is made.
Propionibacteria can use both inorganic and organic nitrogen sources, and their requirements for amino acids vary. Most strains require biotin. Cultures for cheese manufacture are grown on complex media, including hydrolyzed protein and yeast extract with lactic acid as a carbon source (Glatz, 1992).
Propionibacteria grow on lactic acid produced during cheese fermentation. Lactate is oxidized to pyruvate, which then is either converted to acetate and carbon dioxide or propionate. Carbon dioxide forms the large eyes found in Swiss and similar types of cheese, and other metabolic products, including amino acids and fatty acids, contribute to flavor of these cheeses.
Table 3 Differentiation of Propionibacterium spp. Associated with Dairy Products Characteristic Pr. freudenreichii Pr. jensenii Pr. thoenii Pr. acidipropionici
Sucrose Maltose Mannitol
Rhamnose Nitrate reduction ß-hemolysis Colony color
Red-brown Cream to orange-yellow
Source: Cummins and Johnson, 1984.
Brevibacterium cells are aerobic, gram-positive, pleomorphic rods that grow on the surface of surface-ripened varieties of cheese. The species most often isolated from these cheeses is B. linens. B. linens produces a yellow-orange carotenoid pigment that colors the surface of the cheese. Color formation is enhanced by exposure to light. Older cultures are primarily coccoid, but slender rods are produced in exponential growth. B. linens does not use lactose or citrate but can grow on the lactate produced during cheese manufacture. It also grows best at neutral pH, so it does not grow well on the cheese surface until lactic acid is neutralized or metabolized by yeasts or micrococci. Surface-ripened cheeses are surface salted, and B. linens, like yeasts and micrococci, grows well at high salt concentrations. B. linens is highly proteolytic with the ability to degrade whey proteins and casein (Fringa et al., 1993; Holtz and Kunz, 1994). The ability of B. linens to degrade amino acids to ammonia and methionine to methanethiol is partially responsible for production of strong flavors and odors during surface ripening of cheese. Other volatile compounds produced by B. linens that contribute to the typical flavor of surface-ripened cheese include butyric acid, caproic acid, phenylmethanol, dimethyldisulfide, and dimethyltrisulfide (Jollivet et al., 1992). B. linens grows well in media containing hydrolyzed protein, glucose, yeast extract, potassium phosphate, and magnesium sulfate (Haysahi et al., 1990).
The genus Enterococcus includes the Lancefield group D (fecal) streptococci, Streptococcus faecalis and S. faecium, as Ent. faecalis and Ent. faecium. Since reestablishing the genus in 1984, 9 species has been transferred from the genus Streptococcus and 10 new species have been added (Stiles and Holzapfel, 1997; Klein et al., 1998). They are gram-positive, catalase-negative cocci, produce L(+) lactic acid homofermentatively from glucose, and also derive energy from degradation of amino acids. They have a phosphoenolpyruvate phosphotransferase (PEP-PTS) system for uptake of lactose and other carbohydrates, including gluconate.
Enterococci are used as food safety indicators and have a possible involvement in foodborne illness. Enterococci are also used as starter cultures in some southern European cheeses. In addition, they are commercially available as pro-biotics for prevention and treatment of intestinal disorders. Among enterococci only Ent. faecalis and Ent. faecium are important as probiotics. They are readily differentiated by fermentation of arabinose and sorbitol and by their growth temperatures (Klein et al., 1998).
The genus Bifidobacterium is in the family Actinomycetaceae. Bifidobacteria produce lactic and acetic acids in the ratio of 2:3. They have the enzyme fructose-
6-phosphate phosphoketolase which is lacking in lactic acid bacteria. Also, the high G+C content of their DNA (55-57 mol%) and their phylogenetic relat-edness place them in the actinomyces subdivision of gram-positive bacteria. The 29 species exhibit major morphological differences (Stiles and Holzapfel, 1997). The taxonomy and nomenclature of Bifidobacterium is still evolving, and many probiotic cultures now in use do not have the appropriate species designation. Since biochemical reactions are not always useful to classify strains isolated from dairy products, only polyphasic taxomony, which is a combination of phe-notypic and genomic traits, is able to differentiate species (Kien et al., 1998). The natural habitat of bifidobacteria is the intestinal tract. They can also be found in sewage, vaginal microflora, and dental caries. The most important species of Bifidobacterium for probiotic application are B. longum, B. bifidum, and B. ani-malis.
The different enzymatic capabilities of bifidobacteria strains make it difficult to select a single medium for all species (Marshall and Tamime, 1997). Technological selection criteria for bifidobacteria strains to be used as probiotic microorganisms include capability of growing to high cell density in inexpensive media, robust to culture concentration, and the capability of being harvested, frozen or freeze dried with cryoprotection. In addition, the culture must retain its viability and properties throughout the shelf life of the product. Medicoscientific criteria for selection include gastric transit tolerance, small intestinal transit tolerance, bile salt tolerance, limenal growth and persistence, epithelial adhesion, epithelial growth and persistence, coaggregation ability, and antimicrobial production and susceptibility (Charteris et al., 1998).
Bifidobacteria grow poorly in milk; possibly because of the lack of small peptides and free amino acids. Some strains exhibit better growth when milk is supplemented with casein hydrolysate or yeast extract. Strains reported to grow well in milk may be stimulated by naturally occurring growth factors such as specific casein derivatives or oligosaccharides (Marshall and Tamime, 1997).
Because of the possible role of bifidobacteria in stabilizing the digestive system of humans, much attention has recently been given to incorporation of this species into dairy products. In yogurt, they are usually used in combination with normal yogurt bacteria because of their slow acid production. However, postproduction acidification and the possibility that they are inhibited by antimicrobial compounds produced by Lb. delbrueckii subsp. bulgaricus could pose problems for their survival. Although many bifidobacteria are acid sensitive, some strains survive at pH values as low as 4. Variations in survival are affected by storage temperature, the initial number of bacteria, storage time, and strain tested. In cheese, bifidobacteria persist in moderately high numbers in spite of adverse salt content and storage temperature. Generally, bifidobacteria strains exhibit diverse responses to adverse conditions, so appropriate strain selection is very important.
Penicillium spp. are molds in the class Hyphomycetes in the division Deuteromy-cota. Molds in this class produce conidia directly on mycelium or on conidio-phores. The conidiophores of Penicillium spp. arise erect from the hyphae and branch near the tip to produce a brush-like ending (Beneke and Stevenson, 1987). Two groups of Penicillium spp. are used in cheese manufacture, the white mold (P. camemberti Thom, formerly two species, P. caseicolum and P. camemberti), which grows on the surface of Camembert, Brie, and similar varieties; and the blue mold (P. roqueforti, formerly P. roqueforti var. roqueforti), which grows in the interior of blue-veined cheeses such as Roquefort, Gorgonzola, and Stilton. P. camemberti is closely related to P. commune, a common cheese contaminant that produces various toxins (Frisvad and Filtenborg, 1989), whereas P. camem-berti produces only one mycotoxin, cyclopiazonic acid. P. roqueforti is closely related to P. carneum (formerly P. roqueforti var. carneum), a producer of the mycotoxin patulin, and P. paneum (formerly P. roqueforti var. carneum), a producer of patulin and the mycotoxin botryodiploidin (Boysen et al., 1996).
Many complex media are available to cultivate different genera of lactic acid bacteria. However, only a few of them are considered to be selective. Table 4 lists media commonly used to enumerate dairy starter bacteria.
Different means are used to develop selective media which are based on biochemical characteristics (oxygen sensitivity, antibiotic resistance, acid production, fermentation patterns), and bioproducts of the enumerated species. The same medium can be used to enumerate selectively a particular species by changing the incubation temperature (M17 at 37°C for S. thermophilus and 25°C for Lacto-coccus) or by changing the pH (MRS at 5.5 for selective enumeration of Lb. delbrueckii ssp. bulgaricus). Some ingredients are added to inhibit growth of other species. For example, sodium azide makes Elliker agar more selective for lactic acid bacteria. Also, media used to enumerate bifidobacteria are characterized by the presence of substrates, which lowers the redox potential (cysteine, cystine, ascorbic acid), antibiotic, and/or a single carbon source to inhibit lactic acid bacteria. Vacomysin is added to a Leuconostoc medium to inhibit Lactococcus and
Table 4 Media Used for Enumeration of Dairy Starter Cultures
Lactic acid bacteria S. thermophilus
Lb. delbrueckii subsp. bul-
garicus Lb. acidophilus
Leuconostoc ssp. Yeasts and molds Enterococci Lactococci
Differentiate between rods and cocci in yogurt starter
Differentiate between homo- and heterfermenta-tives
Differentiate between Lb. acidophilus, Bifidobacterium spp., S. ther-mophilus and Lb. del-brueckii subsp. bulgaricus
Differentiate between Lac-tococcus lactis subsp.
Elliker (lactic) agar
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