A. Metabolism, Activity, and Reliability
The currently employed food fermentations rely on only a few main metabolic pathways:
Anaerobic alcoholic fermentation, which converts carbohydrates to alcohol and CO2
Lactic fermentation, which converts carbohydrates to lactic acid
Aerobic acetic acid fermentation, which converts alcohol to acetic acid
The main end products—alcohol and organic acids—are responsible for the primary preservation of the foods, and they also contribute significantly to the flavor of the fermented products. Secondary metabolic pathways do, however, create a large variety of flavors and textures, yielding a diversity of products (11). To satisfy their need for nutrients, microorganisms are able to produce hydrolytic enzymes such as proteases, peptidases, lipases, glucanases, amylases, and so forth. In addition to satisfying the microorganism's needs, these enzymes also have the potential to generate or degrade strongly flavored products (19-23). Enzymatic reactions are very important in ripened meat products and cheese. The substrate specificity of enzymes varies considerably among microbial species and strains, and this is an important source of the diversity of starter cultures. The accumulation profile of small flavored metabolites such as diacetyl, acetaldehyde, acetate, and formate also contributes significantly to the diversity of cultures. The balance between these metabolites is determined by the precise metabolic route leading from the carbohydrate to main end products, as well as the utilization of alternative electron acceptors (15). Not only generation of flavor, but also generation of gas and texture, is dependent on the subtle differences between the metabolic routes of the cultures. These differences are responsible for the presence or absence of eyes in cheeses, bubbles or not in fermented milk, and so forth.
A suitable metabolism is, as just described, the primary performance parameter for a starter culture, but next in importance is speed and reliability. The optimization of the fermentation speed involves reducing the lag phase, increasing the growth rate, and increasing the metabolic activity of the cells. Reliability is obtained by selecting strains with low sensitivity to environmental factors that can be encountered in a particular fermentation. A general problem in large-scale fermentations is bacteriophage attack of the starter culture. This has been a particular problem in the cheese industry due to the repeated use of open vats, which cause severe losses. Considerable research efforts have been directed toward the protection of Lactococcus lactis from attacking phages. These efforts have been successful in generating new fundamental knowledge about bacteriophages and in generating new bacteriophage resistance mechanisms (24,25).
A general preservation effect is obtained by most food fermentations from the accumulation of organic acids and alcohols concomitantly with the reduction in free sugar levels, depletion of oxygen, and lowering of the pH (26). Cultures with much stronger preservation effects have been identified and in most cases have been found to produce antimicrobial bacte-riocins (12). Lactobacillus reuteri is an interesting exception because its antimicrobial substance, reuterin, is a low molecular weight metabolite, 3-hydroxypropionaldehyde (27). The first bacteriocin, nisin, was discovered about 70 years ago. Nisin is produced by strains of Lactococcus lactis, and the molecule is a small peptide containing unusual amino acids due to posttranslational modifications (28). Nisin has been in practical use as a food preservative for more than 50 years, and its use is approved in most countries. A large number of bacteriocins have been characterized from lactic acid bacteria and are classified into three groups based on their structural differences (12). The bacteriocins share a common mode of action in their ability to form pores in the membrane of the target bacteria; the molecular aspects of the formation of pores have been well characterized, particularly for nisin (29,30). The ability to produce bacteriocins is quite common among microorganisms isolated from fermented foods, and the consensus among all studies is that this property is beneficial and safe (12,14,31). It is, therefore, to be expected that a number of bioprotective cultures will be introduced into the market.
The beneficial effect of lactic acid bacteria on human health was described by Metchnikoff almost a century ago (32). Several studies have substantiated these positive health effects, which was later named the probiotic effect. The currently used definition of probiotics is as follows: ''live microorganisms, which when consumed in adequate amounts, confer a health effect on the host'' (33). It has, however, turned out to be difficult to identify and prove the mode of action for probiotics (34,35). The bacterial flora of the digestive tract is an extremely complex ecosystem consisting of numerous bacterial species (36-38). The intestinal microbial flora is necessary for the normal function of the digestive system. Elimination or severe perturbations of the flora leads to diarrhea or constipation; therefore, the maintenance of healthy bacterial flora is desirable (35,36). In absence of a well-defined mode of action for probiotics, practical criteria for selecting probiotic strains have been formulated (34,39-41). The main requirements are acid and bile stability, antagonism toward pathogenic bacteria, safety in use, and clinical documentation of the health effects.
V. COMMERCIALLY AVAILABLE STARTER CULTURES A. Production of Starter Cultures
The propagation of microorganisms on an industrial scale is a central and most obvious part of the production of starter cultures. Depending on the product, industrial scale can range from laboratory propagation in flasks and agar plates to fermentors of hundreds of cubic meters in size. Except for a few of the mold cultures produced by sporulation on solid media, most cultures are produced by liquid submerged fermentation. Aerobic fermentations are used for the production of yeast and aerobic flavor cultures; however, the majority of the bacterial cultures are produced by anaerobic fermentations. Less obvious and therefore probably more important for the quality of the culture products are the procedures occurring upstream or downstream relative to the fermentation. Among the upstream processes, the most important ones are those that secure the identity and purity of the microorganisms produced. In order to eliminate the risk of a gradual change of the product over time due to genetic instability of the microorganism, the internal production of inocula must be organized so that each batch has the same ''molecular'' age (42). The identity of the inoculum must also be verified, preferably by DNA fingerprinting methods
The procedures downstream from the fermentation are designed to increase the cell density, to preserve the microorganisms, and to package the products in a format allowing convenient storage, distribution, and use. The cell density can be increased by centrifu-gation or ultra filtration. Depending on the desired format of the product and the fragility of the microorganism, the product can be packaged in liquid, dry, or frozen form. The packaging material must be designed to protect the microorganisms from excessive heat, moisture, and light. The actual sensitivity to these factors can vary considerable among different culture types, but also between different formulations of the same culture.
Typical production processes for starter cultures have been described by Hoier et al.
(44). Culture producers use similar principles for quality assurance and HACCP (hazard analysis critical control point) in the production processes (45).
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