2

Microorganisms
Egon Bech Hansen
Danisco A/S, Copenhagen, Denmark

I.

INTRODUCTION

The aim of this chapter is to give an overview of microorganisms used in the production of fermented foods. More than 50 species of microorganisms are frequently used in food production, and therefore this chapter will focus mainly on common aspects of the use of microbial cultures, rather than on the dierences between the cultures and species. The chapter is intended as a guide for the practical oriented food engineer on how to apply microorganisms in the production of industrially produced fermented foods.

II. A.

FERMENTED FOODS Traditional Food Fermentations

Food fermentation has been used for centuries as a method to preserve perishable food products. The raw materials traditionally used for fermentation are diverse and include fruits, cereals, honey, vegetables, milk, meat, and sh. Fermented products encompass, but are not limited to wine, beer, vinegar, bread, soy sauce, sauerkraut, kimchi, pickled olives, dierent fermented milk products, a large number of cheeses, and a variety of sausages. Popular fermented foods are listed in Table 1 together with the raw materials used and the type of culture involved in the fermentation. Fermentation was invented long before the discovery of microorganisms and the mystery of the process is reected in the common origin of the words for yeast and ghost. It was understood that some processes required an inoculum, and the need for this was satised by keeping a sample from the previous production. This procedure is still in use for propagation of sourdough for private use, and also for the production of some artisanal cheeses. For other processes, inoculation was not necessary because naturally occurring microorganisms in the raw materials could, under proper conditions, be a reliable source of the microbial ora. This is the case in the production of raw milk cheeses, wine, sauerkraut, and some fermented sausages. The production of fermented foods and the characteristic qualities of each are described in detail in other chapters of this handbook. Recent comprehensive reviews of fermented foods have been edited by Wood (1).

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

10 Table 1 Fermented Foods and the Required Ingredients Product Beer Wine Vinegar Bread Soy sauce Sauerkraut, kimchi Fermented sausages Pickled vegetables Fermented milks Cheese Raw material Cereals Grape juice Wine Grains Soybeans Cabbage Meat Cucumbers, olives a.o. Milk Milk Starter culture

Hansen

Yeast Yeast, lactic acid bacteria Acetic acid bacteria Yeast, lactic acid bacteria Mold, lactic acid bacteria Lactic acid bacteria Lactic acid bacteria Lactic acid bacteria Lactic acid bacteria Lactic acid bacteria, yeast, mold

B.

Industrial Food Fermentations

With the discovery of microorganisms, it became possible to understand and manage food fermentations. Methods for isolating and purifying microbial cultures became available in the 19th century. Sterilization or pasteurization of the raw materials prior to inoculation with well-dened cultures allowed the fermentation processes to be managed with little variation. The use of dened cultures became the industrial standard in breweries by the 19th century. During the 20th century, the wine, dairy, and meat industries also shifted production procedures toward the use of well-characterized and dened starter cultures. The application of microbiology and process technology resulted in large improvements in the quality of the fermented food products. The quality improvements have been so great that today all signicant production of fermented food is industrial, or at least professionally performed. The small amount of home fermentations conducted in the form of baking, home brewing, and private cheese making usually rely on commercially available yeast and bacterial cultures. The maintenance of the microorganisms diers between the dierent food industries in the sense that some fermentation industries such as breweries and vinegar producers maintain their own strains and inocula. In the dairy industry, as well as in the meat industry and bakeries, cultures are usually obtained from suppliers dedicated to the production of high-quality food ingredients.

III. A.

MICROORGANISMS Yeast, Molds, and Bacteria

A large variety of microorganisms have been employed in food fermentations. Yeast and mold species commonly used are listed in Table 2, and bacterial species are listed in Table 3. The two lists represent a compilation of the species found in commercially available cultures (210, personal communications by H. Heap and M. B. Prevot, 2002) as well as those commonly found in food fermentations (1114). The lists are not complete with respect to species that are only occasionally found in fermented foods. Of the large number of microorganisms listed, a few are exceptionally widely used. The top three are Saccharomyces cerevisiae, Lactococcus lactis, and Streptococcus thermophilus. Saccharomyces cerevisiae is used as bakers yeast, brewers yeast, inoculums for wine fermentations, food and feed additives, and as avor-generating cultures in dairy and meat products. The annual production of bakers yeast is approximately 1 million tons; the

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Microorganisms Table 2 Genus Aspergillus Candida Candida Candida Candida Candida Candida Geotrichum Penicillium Penicillium Penicillium Penicillium Penicillium Saccharomyces Saccharomyces Yeast and Mold Species Commonly Used in Food Fermentations Species oryzae famata kefyr krusei lipolytica Parapsilosis valida candidum album camemberti chrysogenum nalgiovense roqueforti bayanus cerevisiae Application Soy sauce Meat Fermented milk Fermented milk

11

Cheese, fermented milks Cheese, meat Meat Meat Cheese, meat Fermented milks Bakers yeast, brewing, wine-making, cheese, fermented milks, meat, vegetables, and probiotics

volume of the yeast production exceeds the combined production of all other microorganisms by about two orders of magnitude. The bulk production of yeast is a commodity quite dierent from the high value, low volume yeast strains produced for inoculating wine fermentations, and is also quite dierent from all the other inoculants for food fermentations. Lactococcus lactis is the most widely used lactic acid bacterium. L. lactis is used for the production of cheese, butter, buttermilk, and other fermented milks and, to some extent, is used in meat, bread, and vegetable products. Streptococcus thermophilus is the second most-used lactic acid bacteria. It is used in the dairy industry for the production of yogurt and a variety of other fermented milk products, and for the production of several cheeses, most notably mozzarella and pizza cheese. Due to the high acidication activity, S. thermophilus is often used in combination with other lactic acid bacteria to increase the speed of the fermentation. Of the Lactobacillus species, L. delbrueckii and L. acidophilus are used in relatively large volumes, especially in dairy products; L. acidophilus is also used in various probiotic products. Several species of Bidobacterium are used as probiotic cultures in fermented foods and food supplements. The other species listed in Tables 2 and 3 are produced in much smaller volumes, but for some strains the contribution to the nal product is so essential that the value of the culture can be very high although the volume is small. B. Taxonomy

Identication and classication of microorganisms has traditionally been a dicult task due to the large number of dierent microorganisms of relatively uniform cell morphology and colony morphology. The phenotypes used in classical microbiology are often of the +/ type. Such characters are suited for the identication of individual species but dicult to use

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12 Table 3 Bacterial Species Commonly Used in Food Fermentations Genus Acetobacter Bidobacterium Bidobacterium Bidobacterium Bidobacterium Bidobacterium Bidobacterium Brevibacterium Brevibacterium Carnobacterium Carnobacterium Enterococcus Kocuria Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactococcus Lactococcus Species aceti adolescentis animalisa bidum breve infantis longum casei linens divergens piscicola faecium variansb acidophilus alimentarius brevis casei coryniformis crispatus curvatus delbrueckii subsp. bulgaricus delbrueckii subsp. delbrueckii Delbrueckii subsp. lactis farciminis fermentum gasseri helveticus johnsonii keri panis pentosus plantarum reuteri rhamnosus sakei subsp. carnosus sakei subsp. sakei c sanfranciscensis lactis subsp. cremoris lactis subsp. lactis Application

Hansen

Vinegar production Probiotics Cheese, fermented milks, probiotics Cheese, fermented milks, probiotics Probiotics Probiotics Probiotics Cheese Cheese, bioprotection Meat, bioprotection Meat, bioprotection Cheese, fermented milks, meat, vegetables, probiotics, bioprotection Meat Probiotics, cheese, fermented milks, meat, vegetables Meat Probiotics, vegetables, bioprotection Probiotics, cheese, fermented milks, meat, vegetables Cheese Meat Fermented milks, cheese, probiotics Cheese, vegetables Fermented milks, cheese Meat Cheese, probiotics Fermented milks, probiotics Cheese, fermented milks, probiotics, vegetables Fermented milks, probiotics, probiotics Fermented milks Sourdough, bread Meat Bread, meat, wine, vegetables, bioprotection Bioprotection, probiotics Probiotics Meat Meat, vegetables, bioprotection Bread Cheese, fermented milks, bread, meat, vegetables, probiotics, bioprotection Cheese, fermented milks, bread, meat, vegetables, probiotics, bioprotection

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Microorganisms Table 3 Genus Lactococcus Leuconostoc Leuconostoc Leuconostoc Leuconostoc Leuconostoc Leuconostoc Micrococcus Oenococcus Pediococcus Pediococcus Pediococcus Propionibacterium Propionibacterium Propionibacterium Staphylococcus Staphylococcus Staphylococcus Staphylococcus Streptococcus Weissella Weissella
a b

13

Continued Species lactis subsp. lactis biovar diacetylactis carnosum lactis mesenteroides subsp. cremoris mesenteroides subsp. dextranicum mesenteroides subsp. mesenteroides pseudomesenteroides luteus oeni acidilactici damnosus pentosaceus acidipropionici freudenreichii subsp. freudenreichii freudenreichii subsp. shermanii carnosus subsp. carnosus carnosus subsp. utilis equorum xylosus thermophilus confusa halotolerans Application Cheese, fermented milks, bread, meat, vegetables, probiotics, bioprotection Meat, bioprotection Cheese, fermented milks Cheese, fermented milks, vegetables Probiotics Cheese, fermented milks, vegetables

Meat Wine Meat, probiotics, bioprotection Meat, bioprotection Meat Cheese Cheese, probiotics Cheese Meat Meat Meat Meat Cheese, fermented milks, bread, meat, vegetables, probiotics Meat Meat

Bidobacterium lactis is not a separate species but included in B. animalis. Micrococcus varians has been renamed Kocuria varians. c Lacttobacillus bavaricus has been included in L. sakei subsp. sakei.

in organizing species into higher orders of relatedness. The traits traditionally used for identication are cell morphology, Gram staining, growth on various carbohydrates, gas formation, acid production, temperature prole for growth, salt tolerance, amino acid requirements, vitamin requirements, oxygen requirement/sensitivity, hemolysis, and hydrolysis of polysaccharides, proteins, and lipids. At the strain level, methods for discrimination are based on serological tests, phage typing, or very specic biochemical tests. All these traditional tests are still used and important in the microbiology laboratory, particularly those relating directly to the metabolisms exploited for the fermentation process. Identication, classication, and taxonomy of microorganisms have, however, undergone dramatic changes during the past two decades due to the introduction of methods from molecular biology (15). These methods have allowed us to base identication and taxonomy on the properties common to all microorganisms instad of the traits that dier. Several of the fundamental processes in a living cell are shared by all organisms, and one of

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Hansen

these is protein synthesis. The ubiquitous presence of ribosomes allows taxonomists to use their degree of similarity to deduce evolutionary distances between organisms. For practical reasons the RNA molecules of the ribosome are particularly convenient for this purpose (16, 17). The phylogenetic relationships between the species listed in Table 3 are also presented in Fig. 1. A few G+ pathogens, as well as the G Escherichia coli, have been included for reference, whereas a large number of other bacterial species have been omitted for purposes of clarity. It has previously been pointed out that species of lactic acid bacteria do not have a close phylogenetic relationship, but share a common strategy for survival in nutrient-rich environments (15). By inspecting Fig. 1, it is apparent that further changes in the taxonomy of the Lactobacillus-Pediococcus group will be necessary before the taxons can be represented in clades. Fig. 1 also clearly illustrates how small the evolutionary distance can be between benecial and lethal microorganisms.

Figure 1 The phylogenetic relationship between the bacterial species listed in Table 3. The phylogenetic tree was constructed based on the 16S rRNA sequences using the server of the ribosomal database project (http://rdp.cme.msu.edu, 17). The G- species, Escherichia coli, has been included for reference. Also included as references are a few G+ pathogens and spoilage organisms: Mycobacterium tuberculosis, Clostridium tyrobutyricum, Clostridium botulinum, Streptococcus pneumoniae, Enterococcus faecalis, Staphylococcus aureus, and Bacillus cereus.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

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General methods for identication below the species level are now also based on molecular methods. DNA ngerprint methods able to identify individual strains have been developed; one of the most reliable and portable methods uses pulsed eld gel electrophoresis of restriction patterns of entire bacterial chromosomes (18).

IV. A.

PERFORMANCE PARAMETERS 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 productsalcohol and organic acidsare responsible for the primary preservation of the foods, and they also contribute signicantly to the avor of the fermented products. Secondary metabolic pathways do, however, create a large variety of avors 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 microorganisms needs, these enzymes also have the potential to generate or degrade strongly avored products (1923). Enzymatic reactions are very important in ripened meat products and cheese. The substrate specicity of enzymes varies considerably among microbial species and strains, and this is an important source of the diversity of starter cultures. The accumulation prole of small avored metabolites such as diacetyl, acetaldehyde, acetate, and formate also contributes signicantly 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 avor, but also generation of gas and texture, is dependent on the subtle dierences between the metabolic routes of the cultures. These dierences 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 eorts have been directed toward the protection of Lactococcus lactis from attacking phages. These eorts have been successful in generating new fundamental knowledge about bacteriophages and in generating new bacteriophage resistance mechanisms (24,25). B. Biopreservation

A general preservation eect 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 eects

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Hansen

have been identied and in most cases have been found to produce antimicrobial bacteriocins (12). Lactobacillus reuteri is an interesting exception because its antimicrobial substance, reuterin, is a low molecular weight metabolite, 3-hydroxypropionaldehyde (27). The rst 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 modications (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 classied into three groups based on their structural dierences (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 benecial and safe (12,14,31). It is, therefore, to be expected that a number of bioprotective cultures will be introduced into the market. C. Probiotic Effects

The benecial eect of lactic acid bacteria on human health was described by Metchniko almost a century ago (32). Several studies have substantiated these positive health eects, which was later named the probiotic eect. The currently used denition of probiotics is as follows: live microorganisms, which when consumed in adequate amounts, confer a health eect on the host (33). It has, however, turned out to be dicult to identify and prove the mode of action for probiotics (34,35). The bacterial ora of the digestive tract is an extremely complex ecosystem consisting of numerous bacterial species (3638). The intestinal microbial ora is necessary for the normal function of the digestive system. Elimination or severe perturbations of the ora leads to diarrhea or constipation; therefore, the maintenance of healthy bacterial ora is desirable (35,36). In absence of a welldened mode of action for probiotics, practical criteria for selecting probiotic strains have been formulated (34,3941). The main requirements are acid and bile stability, antagonism toward pathogenic bacteria, safety in use, and clinical documentation of the health eects.

V. A.

COMMERCIALLY AVAILABLE STARTER CULTURES 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 asks 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 avor 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

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Microorganisms

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identity of the inoculum must also be veried, preferably by DNA ngerprinting methods (43). 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 centrifugation or ultra ltration. 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 dierent culture types, but also between dierent formulations of the same culture. Typical production processes for starter cultures have been described by Hier et al. (44). Culture producers use similar principles for quality assurance and HACCP (hazard analysis critical control point) in the production processes (45). B. Formats and Formulations of Cultures

Food fermentations can be inoculated either directly with a highly concentrated starter culture obtained from the supplier, or they can be inoculated from a bulk starter propagated locally. The choice between the two process types will be inuenced by a number of factors: the number of dierent fermented products produced in the same factory, degree of automation, presence of expertise in microbiology and, nally, the economics. The highest level of safety and exibility is achieved by direct inoculation of the culture. In addition to the choice between direct inoculation or bulk starter, there are choices between dierent culture formulations. The usual options are fresh, dried, or frozen, but their availability diers between suppliers and products. Bakers yeast is generally inoculated directly into the dough without propagation in the bakery. Yeast is supplied in both fresh and dry forms; fresh yeast can be obtained as liquid, compressed, or crumbled yeast, and dry yeast either as active or instant yeast (8,9,46). Mold cultures are mainly used as direct inoculants, and the common format is a dry spore preparation (2,5,7). Bacterial cultures are sold as liquid, lyophilized, or frozen cultures. Liquid bacterial cultures will generally lose activity within days, and for direct cultures this format will require a constant supply. Lyophilized or frozen cultures maintain high activity for months or even years, and these formats are ideally suited for global distribution of direct inoculants. Bulk systems for factory propagation of cultures are common for large-scale cheese productions. The cultures used in these systems are supplied from a starter manufacturer in frozen or freeze-dried form, and the media to be used for the propagation is available from the same source (310). C. Quality and Safety of Industrially Produced Cultures

Safety is probably the main reason to buy starter cultures from a commercial supplier rather than propagating local stocks of cultures. The commercial suppliers have the scientic and technical competencies necessary to allow them to maintain purity with respect to crosscontamination as well as protection against harmful contaminants. The preservation of the cultures in lyophilized or frozen form allows the time required to perform extensive quality control, including analyses for activity, identity, and purity. Also, the regulatory competencies are important to assure compliance with the relevant national and international legislation and standards, not only for the culture manufacturer but also for the consumers

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18 Table 4 Range of Starter Cultures from Commercial Suppliers

Bakery Y Alce ASCRC Chr. Hansen CSK CSL Danisco Degussa DSM Gewu rzmu ller Lallemand NZDRI Quest International Rhodia Italy Australia Denmark Netherlands Italy Denmark Germany Netherlands Germany Canada New Zealand United Kingdom France Wine Y B B Meat M&Y B + + + + + + + + + + + + + + + Cheese R + + + + + + M&Y + + + + + Ferm. B + + + + + + + + + + +

Hansen

Other Pro. + + + + + + + + + + + + + + BioP.

+ + + + + +

+ +

(For each food industry the availability of the dierent culture types is indicated by a +.) The culture types are yeast (Y), bacteria (B), mold and yeast (M&Y), bacterial ripening cultures (R), probiotics (Pro.), and bioprotective cultures (BioP.). Ferm. designates fermented milk.

of the food. The safety issue will often lead food manufacturers to have even their own proprietary cultures produced by one of the suppliers. D. Suppliers of Starter Cultures

The following survey contains suppliers engaged in production and development of starter cultures. Manufactures of standard yeast products have not been included. Companies producing starter cultures range in size from small companies supplying cultures only to subscribing food industries to large global companies supplying these and other food ingredients. An overview of the culture ranges is given in Table 4 for small as well as large companies; the range of other food ingredients is outside the scope of this chapter. The country of origin is also included in Table 4, and all companies can easily be found on the worldwide web.

VI.

INNOVATION TRENDS

Innovation in the starter culture industry is stimulated by possibility and need. New possibilities are constantly being opened up by rapid developments in the biological sciences. Our ability to understand complex biological systems has been transformed through the invention of methods to accumulate and analyze large amounts of data. The genomes of more than 100 microorganisms have now been completely sequenced, including several pathogenic bacteria as well as some of the microorganisms used in food fermentations [e.g., Saccharomyces cerevisiae (47) and Lactococcus lactis (48)]. Safe methods for the genetic engineering of food microorganisms have been developed for the most important species,

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and this has opened a wide range of possibilities for the improvement of yeast and lactic acid bacteria metabolism (4954). The practical applications of the modern methods in Europe, however, have been delayed due to public resistance to modern biotechnology. The other factor stimulating innovation is the need for new products. There is a big need for new methods to preserve crops after harvest, to reduce spoilage before consumption. This need is very strong in the less developed world, but also in the highly developed countries do we need better methods to extend shelf life and avoid spoilage. Food fermentation and bioprotective cultures can solve some of these problems. Probiotic cultures with specic health benets, with dened modes of action, is also an area where the market would welcome new products. These two examples are specic areas under the more general need for new cultures or new culture formulations in order to expand the use of benecial microorganisms in food.

ACKNOWLEDGMENTS I am grateful to Dr. Howard Heap (New Zealand Dairy Research Institute), Dr. Claudio Rottigni (Centro Sperimentale del Latte), Dr. Michela Bianco Prevot (Alce), Dr. Francette Hamaide (Degussa), Dr. Odile Conan (CSK food enrichment), Dr. Gae tan Limsowtin (ASCRC), Knud Vindfeldt (Chr. Hansen A/S), and Danny ORegan (Danisco A/S), for providing information on product ranges and bacterial species in commercial use.

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18.

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

38. 39. 40. 41. 42. 43.

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Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.

Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.