Of Microbial Strains Isolated from the Dairy Environment

Cultivable microflora of milk and dairy products is mainly represented by LAB (Lactobacillus, Streptococcus, Enterococcus, Lactococcus, Leuconostoc, Wiessella and Pediococcus). However, strains of other genera such as Propionibacterium, Staphylococcus, Corynebacterium, Brevibacterium, yeasts and molds can also occur. Today (November 2006; see also www.bacterio.cict.fr), the genus Lactobacillus contains 119 species, the genus Streptococcus 67 species, the genus Enterococcus 34 species, the genus Lactococcus 5 species, the genus Leuconostoc 14 species, the genus Wiessella 11 species, the genus Pediococcus 11 species and the genus Propionibacterium 13 species. The main microbial species and their occurrence in dairy products are highlighted in Table 2.1.

The problems of traditional identification methods, even when based on miniaturized easy-to-handle kits or devices, make their use difficult for a reliable identification or biochemical typing of microbial taxa from food.

For these reasons, significant efforts have been made to develop alternative identification methods combining speed, reliability and low cost. These criteria are met by methods based on molecular rather than phenotypic traits. The greatest advantage of DNA-based identification techniques is that these methods focus on the unique nucleic acid sequence of the microorganisms rather than on the phenotypic expression of products that are encoded by the respective genes. Polyphasic taxonomy, however, combines pheno- and genotypic information and forms the basis for current systematic bacteriology.

Moreover, new microbial species are continually being classified, making further identification tools necessary. Therefore, molecular methods have been increasingly used in order to simplify characterization procedures, to provide rapid and reliable identification, or to validate phenotypically determined taxa. Indeed, thanks to the results of the application of molecular tools, in the course of 2006 new species of LAB were described in the genus Lactobacillus (Aslam, et al. 2006; Konstantinov, et al. 2006; Osawa, et al. 2006; Rodas, et al. 2006; Vancanneyt, et al. 2006a), Enterococcus (Carvalho, et al. 2006; Svec, et al. 2006), Streptococcus (Glazunova, et al. 2006), Leuconostoc (Chambel, et al. 2006) and Pediococcus (Franz, et al. 2006). By contrast, in the same period, some species of Lactobacillus (Naser, et al. 2006a-b; Dellaglio, et al. 2006; Felis, et al. 2006), Enterococcus (Naser, et al. 2006c) and Leuconostoc genera (Vancanneyt, et al. 2006b) were re-classified.

The primary objectives of food microbiological analysis are the control of food quality, food preservation, evaluation of starter culture efficiency, and monitoring of particular species/strains during manufacturing. With reference to the development of starter cultures, with consistent and predictable performance, it is widely recognized that extensive characterization of the strains and more detailed knowledge of their physiology, metabolism and genetics are required. Moreover, the increasing number of available commercial strains used as starters requires reliable methods to accurately differentiate strains at both species and biotype levels in pure and mixed cultures in order to defend rights and eliminate risks of confusion during their use.

The taxonomic level of microbial discrimination depends on the sensitivity of the technique used and may range from genus (or species) to strain level (sub-typing or strain typing). The ability of a molecular typing system to discriminate among genetically unrelated isolates is a reflection of the genetic variation seen in the chromosomal DNA of the bacterial species. Usually this variation is high, and differentiation of unrelated isolates can be accomplished using any of a variety of techniques. However, often technologically important traits of dairy microorganisms are not uniformly distributed within a species. Thus, the most important biotypes are often a smaller subset of the many strains that constitute a species. As a consequence, this subset may exhibit relatively little genetic diversity, and it can be difficult to differentiate among strains even with molecular techniques.

Type-ability refers to the ability of a technique to assign an unambiguous result (type) to each isolate. Non-type-able isolates are more common with phenotypic methods, but can also occur with genotypic methods. The reproducibility of a method means the ability to yield the same result upon repeat testing of a bacterial strain. Poor reproducibility may reflect technical variation in the method or biological variation occurring during in vivo or in vitro passage of the organisms to be examined. The discriminatory power of a technique refers to its ability to differentiate among unrelated isolates, ideally assigning each to a different type. In general, phenotypic methods have less discriminatory power than genotypic methods. Most molecular methods require costly material and equipment, but are relatively easy to learn and are applicable to variety of species. On the other hand, phenotypic methods also involve costs in labor and material and are restricted to a few species (sero-typing, phage-typing). Characteristics of some typing systems are reported in Table 2.2.

Although the classical phenotype-based (biotyping) methods are still of importance for daily routine analyses, genotypic methods have increasingly contributed to the in-depth characterization of microorganisms and their differentiation. It may be assumed that the combination of different fundamental and advanced methods

Table 2.2 Characteristics of Some Systems for Typing of Dairy Bacteria

Typing systems

Proportion of strains typeable

Reproducibility

Discrimination Power

Ease of Interpretation1

Ease of Performance

Cost (Time and money)

Phenotypic biotyping

All

Poor

Poor

Moderate

Easy

High

Antimicrobial susceptibility

All

Good

Poor

Easy

Easy

Moderate

patterns

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