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3. Savat-Brunaud et al., 1997

Matalan and Sandine, 1986

McDonald et al., 1987

Ghoddusi and Robinson, 1996

Reddy et al., 1972

some Lactobacillus spp. On the other hand, nonselective media are required to enumerate injured cells; for example, lactobacilli that survive milk pasteurization used for cheese manufacture. Many different media were also developed to assist differentiation of species or subspecies. Differentiation is usually based on colony morphology, which is affected by the interaction between bacteria and medium.

D. Desirable Properties of Lactic Cultures

Properties desired of lactic cultures for industrial use may differ from those found in typical wild-type microorganisms. For example, most dairy fermentations require rapid acid production and the lack of off-flavor production, whereas wildtype organisms are often slow acid producers and produce such off-flavors as fruity, bitter, and malty. Buchenhuiskes (1993) summarized selection criteria for lactic acid bacteria to be used for food fermentations. These include (1) lack of pathogenic or toxic activity (e.g., production of biogenic amines), (2) ability to produce desired changes, (3) ability to dominate competitive microflora, (4) ease of propagation, (5) ease of preservation, and (6) stability of desirable properties during culturing and storage. Specific properties desired in a dairy starter culture depend on the product being produced.

1. Cheddar Cheese

The four main selective criteria for Cheddar cheese cultures are rapid acid production, bacteriophage resistance (see Sec. V), salt sensitivity, and ripening activity (Strauss, 1997). Rapid acid production should occur at a steady rate throughout curd making. This ensures suppression of undesirable microflora, timely cheese manufacture, and the presence of sufficient ripening enzymes from starter microorganisms. Rapid lactose fermentation in lactococci is associated with the presence of a phosphoenol pyruvate-dependent phosphotransferase system (see Chap. 7 for discussion of acid production).

In Cheddar manufacture, salt is added after most of the desired acidity has developed. However, some acid-producing activity is still needed after salting to ensure that all lactose is metabolized. Residual lactose can serve as a substrate for salt-tolerant organisms such as heterofermentative lactobacilli that produce gas and undesirable flavors (Olson, 1990). Growth of starter microflora after salt addition also produces a low oxidation-reduction potential that has a beneficial impact on flavor development and inhibits some spoilage microorganims.

Ripening activity is related to production of proteases and other enzymes. These enzymes must be produced in sufficient quantity to develop the typical Cheddar flavor without off-flavors. Peptidase activity is more important than proteinase activity. In fact, starter culture proteinases are associated with development of a bitter flavor (Visser et al., 1983). Cheese made using 45-75% proteinase-negative cells developed less bitter flavor than cheese made using proteinase-positive cultures (Mills and Thomas, 1980). However, proteinase-negative strains cannot use proteins, so their growth in milk is limited. Starter culture peptidases hydrolyze peptides (including those with bitter flavor) produced by the action of rennet, and, in combination with other microbial enzymes, produce a chemical environment conducive to development of the typical Cheddar flavor. Starter cultures for Cheddar cheese can include strains that specifically enhance ripening but take little or no part in initial acid production (Trepanier et al., 1991). (See Chapter 7 for additional information on starter culture protease systems.)

2. Mozzarella Cheese

Cultures for mozzarella cheese manufacture are combinations of S. thermophilus and either Lb. delbrueckii subsp. bulgaricus or Lb. helveticus. American-style mozzarella is manufactured for use as a food ingredient, especially on pizza. The starter culture contributes to functional properties related to this use, such as stretchability and heat-induced browning. The typical starter culture for American mozzarella manufacture has a 1:5 rod to coccus ratio (McCoy, 1997). This results in rapid initial acid production (by the streptococci) and shortens make time. Lactobacilli produce acid late in manufacture, and are much more proteolytic than streptococci. Proteolysis during storage increases meltability and decreases stretchability of cheese (Oberg et al., 1991a, 1991b). Rod to coccus ratio only slightly influences textural changes during storage (Yun et al., 1995); level of initial inoculum has a greater influence on texture.

Hassan and Frank, (1997) found that capsule-forming nonropy lactic cultures can mimic some of the physical properties of fat in cheese curd. When used as starter cultures, the capsule-forming strains significantly increased water retention by low-fat mozzarella cheese (Perry et al., 1997, 1998).

Starter culture also affects color development during cooking. Many ther-mophilic cultures use only the glucose portion of the lactose molecule, excreting galactose (see Chap. 7). High-browning cheeses contain nearly five times more galactose than low-browning cheeses (Matzdorf et al., 1994). If low-browning cheese is desired, galactose-utilizing cultures such as Lb. helveticus can be used. Using Lb. helveticus instead of Lb. delbrueckii subsp. bulgaricus results in mozzarella cheese with lower galactose levels, improved melting, and decreased make time (Oberg et al., 1991a). Excessive heat during stretching (curd temperature >66°C) can inactivate starter culture enzymes and reduce galactose metabolism and proteolysis during storage (Chen et al., 1994).

3. Swiss Cheese

Starter cultures for Swiss cheese manufacture must survive the high temperatures used in its manufacture (50-52°C). The starter culture is also responsible for development of the typical Swiss cheese flavor and eye formation. The typical

Swiss cheese starter culture consists of S. hemophilus, Lb. helveticus, and P. freudenreichii subsp. shermanii. Mesophilic lactococci are sometimes added to increase acid production early in manufacture. A consistent rate of acid production by the starter is important, because more rapid acid production results in lower moisture content (Turner et al., 1983). Lactose fermentation occurs primarily during the first 24 h of manufacture. Streptococci initially predominate, using lactose and excreting galactose. Subsequent growth of lactobacilli is required for complete utilization of galactose (Hutkins et al., 1986). If all residual sugars are not used, defects from growth of gas-forming microorganisms or brown pigment formation can occur (Harrits and McCoy, 1997).

Propionibacteria grow on lactate produced by the lactic culture, converting it to carbon dioxide, propionic acid, acetic acid, and small amounts of other compounds. Propionibacteria can reach 109 cfu/g and use more than 50% of the lactate at the center of the cheese (Fryer and Peberdy, 1977). Swiss cheese is ripened at 21 °C for eye formation and then aged at 10°C for flavor development. Therefore, the Propionibacterium culture should grow well at 21 °C but not at 10°C (so the eyes do not split) (Harrits and McCoy, 1997). A predictable rate of gas formation at 21 °C is required, because too rapid gas formation results in split eyes (Hettinga et al., 1974).

High-moisture baby Swiss is manufactured using lower cooking temperatures (approximately 40°C) and therefore is produced, not with thermophilic cultures, but with heat-tolerant lactococci. Propionibacteria are still used for eye formation.

4. Cultured Buttermilk and Sour Cream

Cultures for buttermilk, sour cream, and similar products must both acidify the substrate and produce flavor and aroma compounds. Citrate-fermenting bacteria such as Leuc. mesenteroides subsp. cremoris or Lc. lactis subsp. lactis var. diace-tylactis are combined with Lc. lactis subsp. lactis or Lc. lactis subsp. cremoris. Citrate fermentation is discussed in Chapter 7. Diacetyl, the major aromatic compound in these products, can be reduced to acetoin by diacetyl reductase. Cultures should be selected that are low in diacetyl reductase activity. Acetaldehyde is often produced during fermentation, giving the product an undesirable ''green apple'' or yogurt flavor. Leuconostocs (but not lactococci) can metabolize acetal-dehyde to ethanol, with a resulting flavor improvement (Peterson, 1997). Exo-polysaccharide-producing starter cultures might improve the physical properties of low-fat sour cream.

  1. Yogurt a. Acidification Yogurt is made using a combination of S. thermophilus and Lb. delbrueckii subsp. bulgaricus. These organisms grow in a cooperative relationship resulting in rapid acidification. The presence of lactobacilli stimu lates growth of the more weakly proteolytic S. thermophilus, because lactobacilli liberate free amino acids and peptides from casein (Rajagopal and Sadine, 1990). S. thermophilus, in turn, stimulates growth of Lb. delbrueckii subsp. bulgaricus; possibly by removing oxygen, lowering pH, and producing formic acid and pyruvate (Radke-Mitchell and Sandine, 1984). Strains can be selected for the degree to which their growth depends on the presence of other microorganisms (Veda-muthu, 1994). Yogurt may also contain Lb. acidophilus or other nutritionally beneficial cultures. The most important characteristics for yogurt cultures are (1) rapid acidification, (2) production of characteristic balanced flavor, and (3) ability to produce the desired texture. As with other thermophilic rod-coccus dairy fermentations, initial acidification is from growth of S. thermophilus with lactoba-cilli growing later in the fermentation. Excessively rapid acidification can result in overacidification and a harsh flavor. Acidification of yogurt is controlled by refrigeration, but the culture may continue to acidify slowly at cold temperatures.
  2. Flavor The ideal yogurt flavor is a balanced blend of acidity and acetal-dehyde. This is achieved through culture selection, balance of rod to coccus ratio, and fermentation control. The main source of acetaldehyde is from conversion of threonine to acetaldehyde catalyzed by threonine aldolase of Lb. delbrueckii subsp. bulgaricus (Hickey et al., 1983). Lactobacilli, such as Lb. acidophilus, which produces alcohol dehydrogenase, convert acetaldehyde to ethanol (Marshall and Cole, 1983). Therefore, yogurt produced with Lb. acidophilus does not have a typical yogurt flavor. In addition to acetaldehyde, yogurt cultures produce diacetyl, acetoin, acetone, ethanol, and butanone-2 (Beshkova et al., 1998). Volatile saturated free fatty acids such as acetic, butyric, and capric may also contribute to the flavor of yogurt. Although yogurt cultures are considered to be weakly proteolytic, they cause significant proteolysis in yogurt (Tamime and Deeth, 1980) which can lead to the development of bitterness.
  3. Texture The texture of yogurt results from a complex interaction between milk proteins, acid, and exocellular polysaccharide produced by the starter culture. Important physical properties include firmness, smoothness, viscosity, and gel stability (susceptibility to syneresis). The starter culture can influence each of these properties by production of exopolysaccharides.

Yogurt cultures produce exopolysaccharide in a ropy or capsular form (Ariga et al., 1992). Capsular polysaccharides are formed as a discrete structure surrounding the cell (Fig. 1A) with no apparent interaction with casein (Hassan et al., 1995a, 1995b). Ropy polysaccharides are produced as filaments that are not visualized as discrete structures by light microscopy. Hassan et al. (1996a) classified yogurt cultures into three types: those that do not produce exopolysac-charide, those that produce capsular polysaccharide, and those that produce both capsular and ropy polysaccharide. Cultures that produce only ropy polysaccharide may exist, but an extensive survey has not been reported.

Figure 1 Confocal scanning laser micrographs of encapsulated S. thermophilus. (A) Encapsulated cells in milk visualized using reflected light. (B) pH gradient surrounding encapsulated cells in milk visualized using a pH-sensitive fluorochrome. Dark areas indicate low pH.

Yogurt cultures produce heteropolysaccharides that consist of different sugar residues in a repeating pattern. Their production does not depend on the presence of a specific substrate. In contrast, homopolysaccharides, such as dex-tran produced by Leuc. mesenteroides, consist of one sugar residue type (in this instance, glucose), and a specific substrate is required for their production (in this instance, sucrose). Cerning et al. (1986) found that the heteropolysaccharide produced by Lb. delbrueckii subsp. bulgaricus comprised primarily galactose, glucose, and rhamnose in a molar ratio of 4: 1: 1. On the other hand, S. thermophi-lus capsules are composed of D-galactose, L-rahmnose, and L-fucose in a ratio of 5:2:1 (Low et al., 1998). Garcia-Garibay and Marshall (1991) found evidence that this exopolysaccharide is closely associated with protein and may be better classified as a glycoprotein. The exopolysaccharide of S. thermophilus is composed mainly of galactose and glucose with a small amount of other sugars (Cern-ing et al., 1988). Cerning (1990) stated that there is little agreement as to the precise composition of these polysaccharides.

The influence of ropy polysaccharide on yogurt texture is well documented, but reported effects must be interpreted with caution, because nonropy cultures used as controls were not examined for capsule production until recently. There is general agreement that ropy cultures can benefit yogurt texture by increasing viscosity and gel stability (Cerning, 1990).

However, overproduction of ropy polysaccharide yields a product with an undesirable slippery mouth feel and pronounced ropiness. Capsular polysaccharide cannot be overproduced, because capsule size is limited (Hassan et al., 1995a). Bacterial capsules disrupt the yogurt gel microstructure, producing a softer texture (Hassan et al., 1995b). Encapsulated cultures with no ropy characteristic produce yogurt that is more viscous, structurally more stable, and less susceptible to syneresis than do cultures that do not produce capsules (Hassan et al., 1996a, 1996b). The capsule also slows diffusion of lactic acid away from the cell, causing the cells to stop acid production sooner (Hassan et al., 1995a). This helps prevent overacidification of the yogurt. The pH gradient resulting from encapsulation can be visualized using confocal scanning laser microscopy, as shown in Figure 1B.

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