Starter Culture Microorganisms

A. General Characteristics of Lactic Acid Bacteria

All dairy fermentations use lactic acid bacteria for acidification and flavor production. Although lactic acid bacteria are genetically diverse, common characteristics of this group include being gram-positive, non-spore forming, nonpigmented, and unable to produce iron-containing porphyrin compounds (catalase and cytochrome); growing anaerobically but being aerotolerant; and obligately fermenting sugar with lactic acid as a major endproduct. Lactic acid bacteria tend to be nutritionally fastidious, often requiring specific amino acids, B vitamins, and other growth factors, whereas being unable to use complex carbohydrates.

1. Taxonomy

There are currently 11 genera of lactic acid bacteria, of which four—Lactobacillus, Streptococcus, Lactococcus, and Leuconostoc—are commonly found in dairy starter cultures. A fifth genus, Enterococcus, is occasionally found in mixed-strain (undefined) starter cultures. Important phenotypic taxonomic criteria include morphological appearance (rod or coccus), fermentation endproducts (homofermentative or heterofermentative), carbohydrate fermentation, growth temperature range, optical configuration of lactic acid produced, and salt tolerance (Axelsson, 1993). rRNA sequences are used accurately to determine phylo-genetic relationships among bacteria. This and other genetic methods have led to reorganization of some genera of lactic acid bacteria (e.g., reclassification of lactic streptococci to Lactococcus spp.).

2. Natural Habitat

Lactic acid bacteria are generally associated with nutrient-rich habitats containing simple sugars. These include raw milk, meat, fruits, and vegetables. They grow with yeast in wine, beer, and bread fermentations. In nature, they are found in the dairy farm environment and in decomposing vegetation, including silage. Some species colonize animal organs, including the mouth, intestine, and vagina. They are also part of the normal microflora of the streak canal of the mammary gland. Lactic acid bacteria isolated from natural habitats are often physiologically distinct from their starter culture variants. For example, lactococci isolated from plants ferment lactose slowly, if at all (Chassy and Murphy, 1993).

  1. Characteristics of Starter Culture Genera and Species
  2. Lactococcus

Lactococci (formerly group N streptococci) are the major mesophilic microorganisms used for acid production in dairy fermentations. Although five species are recognized, only one, Lc. lactis, is of significance in dairy fermentations. Lc. lactis cells are cocci that usually occur in chains, although single and paired cells are also found. They are homofermentative; when grown in milk, more than 95% of their endproduct is lactic acid (of the L isomer). Lactococci grow at 10°C but not at 45°C. They are weakly proteolytic and can use milk proteins. They hydrolyse milk casein by extracellular proteinase PrtP. However, all their peptidases seem to be intracellular (Law and Haandrikman, 1997). There are two subspecies, Lc. lactis subsp. lactis and Lc. lactis subsp. cremoris. Differential characteristics for these subspecies are presented in Table 1. Lc. lactis subsp. lactis is more heat and salt tolerant than Lc. lactis subsp. cremoris. A variant of Lc. lactis (Lc. lactis subsp. lactis var. diacetylactis) converts citrate to diacetyl, carbon dioxide, and other compounds. Some lactococci produce exopolysaccharide (Cerning, 1990). These variants are used to produce Scandinavian cultured milks having a ropy texture (viilli, taettamilk, and langmjolk). Another variant of Lc. lactis produces malty off-flavor caused by aldehyde production from amino acids (Morgan, 1976).

2. Streptococcus

The only Streptococcus sp. useful in dairy fermentation is S. thermophilus. This microorganism is genetically similar to oral streptococci (S. salivarius) but can still be considered a separate species (Axelsson, 1993). S. thermophilus is differentiated from other streptococci (and lactococci) by its heat resistance, ability to grow at 52°C, and ability to ferment only a limited number of carbohydrates (Axelsson, 1993). Most dairy products subjected to high temperatures during fermentation (>40°C) are acidified by the combined growth of S. thermophilus and Lactobacillus spp. S. thermophilus has limited proteolytic ability, although it possesses many types of proteolytic enzymes.

3. Leuconostoc

Leuconostoc spp. are distinguished from other lactic acid bacteria by being meso-philic heterofermentative cocci. They do not hydrolyze arginine and require vari-

Table 1 Differentiation of Lactococci Used in Starter Cultures

Characteristic

Lactococcus lactis subsp. lactis cremoris

Acid from Lactose Galactose Maltose Ribose Growth in 4% salt Arginine hydrolysis

Source: Schleifer et al., 1985.

ous B vitamins for growth. Leuconostoc spp. used in the dairy industry produce diacetyl, carbon dioxide, and acetoin from citrate. Some also produce exopolysac-charide (dextran) from sucrose. Only two species of Leuconostoc are associated with dairy starter cultures, Leuc. mesenteroides subsp. cremoris (previously, Leuc. citrovorum) and Leuc. lactis. These are differentiated by their ability to ferment various carbohydrates. Leuconostoc spp. grow poorly in milk; probably because they are adapted to growth on vegetables and roots (Vedamuthu, 1994) and therefore lack sufficient proteolytic ability to grow in milk. Leuc. mesenteroides subsp. cremoris does not produce sufficient acidity in milk to coagulate it, but Leuc. lactis may (Thunell, 1995). In starter cultures, Leuconostoc spp. are combined with lactococci when production of diacetyl and carbon dioxide is desired in addition to acidification. When used in cultured milk starters, they convert excess acetaldehyde to diacetyl, thus reducing undesirable ''green'' flavor (Lindsay et al., 1965). Leuconostoc spp. do not grow well in high-phosphate phage-inhibitory media (Vedamuthu, 1994).

4. Lactobacillus

The Lactobacillus genus consists of a genetically and physiologically diverse group of rod-shaped lactic acid bacteria. The genus can be divided into three groups based on fermentation endproducts. Species in each of these groups can be found in dairy starter cultures, as listed in Table 2. Homofermentative lactobacilli exclusively ferment hexose sugars to lactic acid by the Embden-Meyerhof pathway. They do not ferment pentose sugars or gluconate. These are the lactobacilli (Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lactis, and Lb. helveti-cus) commonly found in starter cultures. They grow at higher temperatures (>45°C) than lactobacilli in the other groups and are thermoduric. Another member of this group, Lb. acidophilus, is not a starter culture organism, but it is added to dairy foods for its nutritional benefits.

Facultatively heterofermentative lactobacilli ferment hexose sugars either only to lactic acid or to lactic acid, acetic acid, ethanol, and formic acid when glucose is limited. Pentose sugars are fermented to lactic and acetic acid via the phosphoketolase pathway. This group includes Lb. casei, which is not usually found in starter cultures but is associated with beneficial secondary fermentation during cheese ripening.

Obligately heterofermentative lactobacilli ferment hexose sugars to lactic acid, acetic acid (or ethanol), and carbon dioxide using the phosphoketolase pathway. Pentose sugars are also fermented using this pathway. These lactobacilli can cause undesirable flavor and gas formation during ripening of cheese. They produce proteinases, endopeptidases, aminopeptidases, dipeptidases, tripepti-dases, and proline-specific peptidases (Law and Haandrikman, 1997). One species, Lb. kefir, is associated with kefir cultures.

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