Engineering and Manufacturing for Biotechnology - Marcel Hofman & Philippe Thonart

Weekers F., Jacques P., Mergeay M and Thonart P.

protecting compounds and growth conditions affect the desiccation-tolerance of the microorganisms. The conditions of storage of desiccated biological materials must be controlled because the nucleic acid damages accumulate during the time of desiccation.

The complexity of the desiccation-tolerance phenomena is related to the complexity of a cell and to the multiplicity of its components. There is not a universal additive that will protect all cells from all desiccation damages, nor there are techniques and conditions that will allow best survival and storage preservation in any case. Each species is a different case.

Quick selection techniques such as resistance to UV radiation exposure can be used to select desiccation-tolerant strains for their technological application.

Acknowledgements

F. Weekers is a recipient of a FRIA (Fonds pour la Formation a la Recherche dans 1'Industrie et 1'Agriculture) grant.

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acceptability and microbiological safety in tropical climates (Haggblade and Holzapfel, 1989).

Since a long time, these interactions of lactic acid bacteria with foods have attracted the attention of scientists and the first research was done on lactic acid fermentation by Pasteur in 1857, followed by the first isolation of a pure bacterial culture, Bacterium lactis, by Lister in 1873. In 1890, Weigmann in Kiel and Storch in Copenhagen introduced, almost simultaneously, the use of starter cultures for cheese and sour milk production. This opened the way for the industrialisation of fermentations with lactic acid bacteria. At about the same time, Elie Metchnikoff at the Pasteur Institute in Paris and other scientists realised the similarity between the food fermenting bacteria and some of the inhabitants of the human intestinal flora and proposed their use in the diet due to health and life prolonging properties. Hence, yoghurt products started to gain popularity in Europe. The development of fruit and flavoured yoghurt in the 1950s helped this product to become even more popular in the western world. Today, a multitude of different fermented dairy and non-dairy foods is commercialised worldwide and the consumer demand especially for fresh refrigerated dairy products is still growing considerably.

2. Classification of lactic acid bacteria

The natural microbial diversity of lactic acid bacteria bears great potential for various applications in food technology and biotechnology. Hence, there is a strong need to develop culture collections, and to classify or categorise the bacteria according to their natural habitat, general properties, their present or past use in foods, and safety issues relevant to man and the environment. The taxonomic classification helps to answer questions like: which bacterial species have a long safety record in food products and are suited for human consumption? And, are there any lactic acid bacteria involved in or known as causative agent for human disease or infections, and hence not suited for consumption? Furthermore, it allows comparisons to be made with bacteria found in (or used for) fermented food and those colonising the human intestinal flora further substantiating the early findings of Metchnikoff.

2.1. THE GROUP OF LACTIC ACID BACTERIA

According to Orla-Jensen (1919), lactic acid bacteria are Gram-positive, non-motile, non-sporeforming bacteria that ferment carbohydrates and higher alcohols to produce lactic acid as the major end product. They comprise different genera and more than 100 different species (Orla-Jensen, 1919; Kandler and Weiss, 1986). Lactic acid bacteria are evolutionary very dispersed comprising microorganisms with different morphologies (coccoidal and rod-shaped), with different optimal growth temperatures (mesophilic and thermophilic conditions), and with different major fermentation pathways. Lactose may be taken up via the phosphoenolpyruvate-sugar phosphotransferase system (PTS) or as free lactose, and glucose may be fermented via the glycolytic (homofermentative) or the pentose phosphate heterofermentative pathways.

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2.2. CLASSICAL BACTERIAL TAXONOMY COMBINED WITH MOLECULAR BIOLOGY

The classical approach to bacterial taxonomy was based on morphological, physiological and biochemical features. Nowadays other characteristics of the cells such as cell wall composition and protein fingerprinting by analysis of the total soluble cytoplasmatic proteins, isoprenoid quinones are included. With the evolution of molecular biology other taxonomic tools became available, such as mol% content of the DNA, pulsed field gel electrophoresis (PFGE), random amplification of polymorphic DNA-PCR (RAPD-PCR), Insertion Sequence (IS) typing, DNA:DNA hybridisation studies and structures and sequence of ribosomal RNA (rRNA). These new tools are especially useful for the identification of lactic acid bacteria which cannot be reliably differentiated by the classical methods, as shown for the species of the

Lactobacillus acidophilus group, comprising of Lb. acidophilus sensu strictol, Lb. amylovorus, Lb. crispatus, Lb. gallinarum, Lb. gasseri, and Lb. johnsonii (Schleifer et al., 1995; Klein et al., 1998). A reliable and fast identification of these organisms is currently only possible with the help of molecular biological methods.

The implementation of molecular biological methods has led to significant changes in the taxonomy of lactic acid bacteria (Schleifer, 1987; Schleifer et al., 1995; Stiles and Holzapfel, 1997; Klein et al., 1998). It has been proposed that the taxonomy and physiology of lactic acid bacteria can only be understood by combining the morphological, biochemical and physiological characteristics with the molecular-based and genomic techniques (Klein et al., 1998). However, the classification of lactic acid bacteria is still under investigation and not without some controversy on the definition of the boundaries between some genera and species (for reviews see: Stiles and Holzapfel, 1997; Axelsson, 1993; Potetal., 1994).

2.3. ISOLATION OF NEW STRAINS OF LACTIC ACID BACTERIA

Lactic acid bacteria are generally found in fermented foods, but also in the gastrointestinal microflora. It is from these habitats that they can be isolated for the use as starter cultures in controlled fermentation processes. An example of the isolation and identification of a new species from cottage industry or spontaneously fermented foods is the new Lactobacillus species, Lb. panis, named after the latin word for bread since it was first isolated from rye sourdough (Wiese et al., 1996). Such species can potentially be further developed into new starter strains for fermentation processes.

However, a major drawback in the isolation of new strains is that many species cannot be cultured in vitro at all. It has been speculated that at present only 10 to 50% of the bacteria from the gastrointestinal tract can be cultured in the laboratory

(McFarlene et al., 1994; Amann et al., 1995; Langendijk et al., 1995; Wilson et al.,

1996). Furthermore, only a fraction of the total culturable and only a few unculturable species are known. The development of novel molecular typing techniques based on DNA technology, by which bacteria can be identified without cultivation, now at least

1 The unconventional abbreviations Lb. and Lc. are used in this text to avoid confusion between the genera

Lactobacillus and Lactococcus, respectively.

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partly overcomes this problem and has greatly facilitated the retrieval of new species (Amann et al., 1990; 1995; Zoetendal et al., 1998). At present, several thousand 16S rRNA sequences of different bacteria are available in genetic databases (Angert et al., 1990; Snel et al., 1994). However, the lack of cultivability restricts their physiological analysis and use in the food technology.

3. Lactic acid bacteria as starter cultures

As outlined above, different members of the lactic acid bacteria are applied as starter cultures for the production of a great variety of foods such as fermented cheese, milk, bread, wine, pickles and meat (Table 1). Important prerequisite for lactic acid bacteria strains used in industrial application are that they must be cultivable and stable on an industrial scale and through the manufacturing processes and storage conditions of the food product over the many years of industrial use. This is especially important for probiotic strains that need to retain their metabolic activity in order to exert the desirable health beneficial effects. Using modern molecular-based tools, industrial foodmicrobiology laboratories are able to characterise and monitor the genetic stability and activity of their starter strains.

3.1. THE ROLE OF LACTIC ACID BACTERIA IN THE FERMENTATION OF MILK

Lactic acid bacteria need to fulfil specific requirements for the transformation of the raw material, e.g. milk, to the final product. These requirements are different depending on the nature of the raw material, the desired end product and the final quality demand.

The role of the lactic acid bacteria in food fermentations can be summarised as: i) generation of flavour, ii) texturing capacities, iii) microbial preservation of the raw material and iv) probiotic, health beneficial properties (Table 2).

The numerous metabolic pathways involved in these functions vary widely. Flavour compounds in dairy products such as yoghurt often result from hydrolysis of milk proteins and lipids and subsequent metabolism of the products, but are equally generated from the bacterial carbon metabolism. The main end product of sugar metabolism, pyruvate, plays a central role since it is converted to metabolites such as lactic acid, acetaldehyde, diacetyl and acetoin which are important for flavour. At the same time, the pH drop caused by the production of lactic acid and other organic acids is responsible for the precipitation of casein and thus necessary for the development of the typical yoghurt texture. Extracellular polysaccharides produced by several lactic acid bacteria have an important impact on texture and viscosity of fermented milk products. Lactic acid bacteria generally inhibit the growth of spoilage and pathogenic bacteria due to the production of lactic acid and ‘natural preservatives’ such as organic acids, hydrogen peroxide and antibacterial peptides, i.e. bacteriocins (Ray and Daeschel, 1992).

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3.2. THE NEW AGE OF STRAIN AND PRODUCT DEVELOPMENT

Several of the early biotechnological processes are still in use, although they are applied today under well-controlled conditions on an industrial scale. To obtain fermentation products of a reproducible and high quality, present large-scale fermentations are initiated by the addition of well-defined lactic acid bacteria starter cultures. Over the last decades, more and more starter cultures were developed for specific product and quality ranges which led to an increased competition in the classical yoghurt market between food companies, local dairies and co-operatives. Therefore the necessity for food companies to further distinguish their products by superior quality and taste, and to offer innovative new products satisfying the consumer needs is increasing. Hence, food companies started to develop proprietary starter cultures to improve their own fermented dairy products.

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In earlier times, new starter cultures were selected by extensive and time consuming screening of large numbers of natural lactic acid bacteria and suitable strains combined by simple trial and error. Today, technological aspects like fermentation and storage conditions, organoleptic influences on taste and texture, as well as health and nutritional aspects of the products and the starter cultures are becoming more comprehensible in molecular terms. Modern microbiology techniques in analytical biochemistry, fermentation technology, and molecular biology allow a more efficient and specific screening of strains or spontaneously occurring mutants in order to identify the one(s) exactly suited for a special purpose. Furthermore, the molecular techniques can also be applied for appropriate genetic improvement of a given natural strain.

4. Improved starter strains – case studies

In the following sections, some results and practical applications in the field of fresh fermented dairy products, including some examples from our own institute, will be presented.

4.1.SELECTION OF NATURALLY IMPROVED STRAINS

4.1.1.Mild, shelf-stable yoghurt

Yoghurt results from the growth association between Streptococcus thermophilus and

Lactobacillus bulgaricus. Both organisms grow in milk where they ferment lactose to lactate, lowering the pH of the product. Upon refrigerated storage of the completed yoghurt for several days (in the supermarkets or at the consumer’s home), the pH may drop further. This so-called post-acidification leads to a gradually increasing acid and bitter taste of the yoghurt, thus degrading the initial organoleptic quality of the product.

S. thermophilus on its own ferments milk into a mild but flavourless product. It is Lb. bulgaricus which mostly contributes to the typical yoghurt flavour, and lowers the pH to values below 4.2 (Oberman, 1985). The approach to limit post-acidification and still produce the yoghurt flavour was to regulate growth and maintenance of Lb. bulgaricus by controlling its energy metabolism. Hence, Lb. bulgaricus starter strains were screened for the presence of spontaneous Lac minus mutants, having no or reduced residual galactosidase activity. Mutants were identified having genetic deletions within the galactosidase gene (lacZ) or expanding beyond the lac region, thereby inactivating a further gene vital for growth in milk, encoding the cell wall bound proteinase (Mollet and Delley, 1990; Germond et al., 1995). Such Lac minus and

Lac Prt minus mutants were not able to grow in milk as single-strain cultures without the supplementation of glucose and peptones. However, if grown in mixed cultures with a lactose fermenting S. thermophilus strain, Lac minus Lb. bulgaricus strains were able to grow despite the absence of glucose. Hence, S. thermophilus provides the mutant

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