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## Über dieses Buch

Aroma biotechnology opens access to natural volatile flavors. Due to the attribute of "naturalness" these aromas are the most valuable ingredients for foods, cosmetics and related products. The author describes biocatalyzed reactions and biogenetic routes leading to aroma and flavor compounds and he depicts in detail their utilisation in industrial scale processes. All the important topics, including de-novo-synthesis, genetic engineering, in vitro rDNA methods, laboratory requirements and techniques, upscaling, product recovery, profitability, and legal aspects are covered. R.G. Berger contributes his own experimental experience to all except one chapter and leads the reader into state-of-the-art aroma biotechnology.

## Inhaltsverzeichnis

### 1. Aroma Compounds in Food

Abstract
The consumption of foods and beverages is inseparably linked to the stimulation of the human chemical senses, odor and taste. The sensation of odor (smell) is triggered by highly complex mixtures of small, rather hydrophobic molecules from many chemical classes that occur in trace concentrations and are detected by receptor cells of the olfactory epithelium inside the nasal cavity. The nonvolatile chemical messengers of the sense of taste interact with receptors located on the tongue and impart, though not limited by polarity or molecular size, four basic impressions only: sweet, sour, salty, and bitter. Also perceived inside the oral cavity, but transmitted to the brain by nonspecific and trigeminal neurons, are pungent, cooling and hot principles.
Ralf G. Berger

### 2. The Roots: Empirical Food Biotechnologies and Formation of Aroma Compounds

Abstract
Our ancestors observed that milk, wet cereal flour, fruit juices, and raw meat, when incubated for some time, underwent changes that led to more stable products (for example, Michel et al., 1992). The resulting foods, altered in texture, color, acidity, gas content, turbidity, and flavor apparently had no adverse effects on human well-being, if consumed in moderation. These very roots of modern biotechnology have evolved from artisan levels into major industries. The present output of the traditional biotechnologies far exceeds the new fermentation products in both volume and product value. According to recent year books the annual biotechnology of antibiotics is worth about 50 bio. US$, while wine and beer production amounted to an estimated 300 bio. US$. A large number of textbooks, encyclopedias and original papers have discussed all the facets of the traditional, fermented foods. This chapter will not recapitulate earlier reviews, but, subdivided under commodity categories, discuss the most recent aroma aspects. The dominating topics will be:
• of the aroma profile of existing fermented products, and
• the possible transfer of existing knowledge and proven technology to novel processes.
Ralf G. Berger

### 3. Why Novel Biotechnology of Aromas?

Abstract
As outlined in the preceding chapter, the products of food biotechnology continue to command a significant portion of the food market. More recently, bioprocesses have been developed for nonvolatile flavor compounds to be used as food additives. Manufactured on an industrial scale are various organic and amino acids, 5-nucleotides, and several types of hexose syrups (Tombs, 1990). On this broad background increasing interest in bioprocesses for the generation of volatile flavors in both academia and industry can be noticed. The multiple and different reasons of this interest are mainly linked with:
• problems of supply from agriculture
• the present legal restrictions
• the high stereochemical requirements, and
• the dramatic advances in cell biochemistry and bioengineering.
Ralf G. Berger

### 4. Laboratory Requirements and Techniques

Abstract
This chapter will provide the beginner with some practical introduction and experience gathered with the laboratory-scale cultivation of aroma producing microorganisms and plant cells. Basic and specific laboratory equipment and the infrastructure required will be described. For a detailed discussion of general biochemical, microbiological, or biotechnological principles a great number of textbooks are available; advice direct from an experienced microbiologist/biotechnologist will help to get started more rapidly.
Ralf G. Berger

### 5. Aroma Compounds From Microbial De Novo Synthesis

Abstract
The aroma compounds of traditionally fermented foods originate, at least in the first phase, from a complex microflora that acts in an only partially understood way on the chemical precursors of a complex food matrix (cf. chapter 2). Most of the classical processes are highly self-regulated and result in constant patterns of volatiles, others depend on narrow windows of the biocatalyst–s properties and manufacturing variables with the imminent risk of off-flavor formation. A concerted biotechnology of aromas requires a defined microbiology and nonfood nutrient media. The following chapter will mainly discuss selected work carried out with modified, but usually not optimized media to illustrate scope and depth of current research. From an application point of view a subdivision of topics according to the chemical structures identified was thought to be preferable.
Ralf G. Berger

### 6. Biotransformation/Bioconversion

Abstract
Many volatile target compounds are not amenable to a microbial de novo synthesis in anything like acceptable yields. Both constitutive or inducable microbial enzymes, however, turn over biotic intermediates and even xenobiotics in single step (biotransformations) or multistep reactions (bioconversions) to products more valuable from a flavor point of view. Biocatalysts can enlarge, degrade, or modify the substrate, thereby supplementing or replacing chemosynthesis. Particularly attractive is the breakdown of complex natural products to volatiles. Recent oil tanker accidents near the Shetlands and in Prince William Sound, Alaska, have unintentionally demonstrated to a broader public the impressive biodegradative capabilities of the indigenous microflora (Venosa et al., 1992).
Ralf G. Berger

### 7. Enzyme Technology

Abstract
In the past decade, biocatalyzed reactions have almost developed into a fashion in synthetic organic chemistry (Faber, 1992). If a given organic molecule is to be modified in a single-step, cofactor-independent reaction, no plausible reason remains for supporting all the other biomass and metabolic energy delivering pathways of an intact cell (cf. Table 3.1). Of the estimated 25 000 enzymes present in nature, about 2800 have been classified, and about 400, mainly hydrolases, transferases, and oxidoreductases have been commercialized. Less than 50 different enzymes are used on a larger industrial scale with a strong emphasis on detergents and food processing. In 1992, the dairy industry and starch/sugar-processing shared 56 mio and 44 mio US$, respectively, of the total sales of 350 mio US$ of technical enzymes in Europe. The vast majority of enzymes in food processing are hydrolases, such as amylases, proteases, pectinases, cellulases, pentosanases, invertase, and lactase. Immobilization techniques, such as inclusion in gels, entrapment in microcapsules, or covalent or adsorptive binding onto solid supports have improved technical aspects of handling, recycling, and long-term stability. Since microbial enzymes have become an integrated part of processes aimed at value-added food, it would seem obvious to propagate their use for the generation of volatiles (Whitaker, 1992).
Ralf G. Berger

### 8. Genetically Altered Catalysts

Abstract
Preceding chapters have dealt with de novo syntheses, conversions and transformation reactions of substantial potential for the biotechnology of aromas. Satisfactory yields and the desired target compound(s) have, however, often not been achieved instantly. This has stimulated interest in classically mutagenized and genetically engineered strains with improved performance. The recombinant DNA technique has dramatically extended possibilities by providing a rapid means of introducing predetermined genetic alterations with high accuracy. A more rapid progress in the field is still impeded by safety considerations, public concern, and legal and administrative (partially self) restrictions. Instead of completely covering all aspects of the emotional discussion, some facts may be remembered:
• Gene transfer is an everyday event in nature. It was even hypothesized that bacterial transformation evolved as a nutrient uptake mechanism, because unrelated DNA is abundant in some natural environments (Redfield, 1993).
• Plasmid harboring cells are ecologically handicapped as compared to the wild strain. Difference in growth rates has been suggested as a diagnostic character (Park and Ryu, 1992).
• Safety testing of food generated with the aid of genetically manipulated strains is emphasized, particularly if the microorganisms are to remain in the consumed product.
Ralf G. Berger

### 9. Plant Catalysts

Abstract
Most of the volatile flavors currently processed by the food industry are directly or indirectly based on the metabolic potential of plants (cf. Sect. 3.1). Various options exist for the deliberate use of this potential by the biotechnologist:
• Crude or partially purified enzymes, if unavailable from a microbial source, can perform transformations of selected substrates according to chapter 6.
• Incubated homogenates bring together previously compartmentalized substrates and enzymes for catabolic reactions.
• Intact parts of plant catalyze anabolic reactions from exogenous substrates.
• In vitro plant cells produce volatiles de novo or by transformation/conversion reactions.
• Genetically altered cells with improved flavor formation capability are multiplied by clonal micropropagation of known overproducing individuals, or obtained by selecting somaclonal variants from in vitro cultures, or obtained by direct transfer of heterologous genes.
Ralf G. Berger

### 10. Bioprocess Technology

Abstract
Type, stability, morphology, and surface properties of biocatalysts used for aroma generation (except plant cells) do not fundamentally differ from those of biocatalysts used in established processes of food and fine chemicals biotechnology. Similarly, the composition, rheology, coalescence and foaming properties of the nutrient media and the physical process parameters of present aroma yielding processes are comparable to data gathered from a variety of prokaryotic and eukaryotic cultivations. As a result, typical design parameters have to be considered for the layout of the reactor, scale-up, and optimization:
• Mass transfer in submerged cultivations has been well studied and proceeds in a three-phase system (microorganisms, liquid bulk phase, gas bubbles). Solid state and immobilized enzyme reactors have been much less investigated.
• Heat transfer, depending on the metabolic waste heat and the biological optimum temperature, can have a negative or positive sign. Hot spots in immobilized or self-aggregated systems are difficult to control.
• Kinetics of the biochemistry (growth, substrate and product concentrations) should be devoid of inhibitory or inactivation effects.
• Physico-chemical parameters (viscosity, equilibria, interfacial interactions) can be crucial and pose optimization problems particularly in integrated processes.
Ralf G. Berger

### 11. Toward an Industrial Application

Abstract
The ultimate goal of process development is to increase the specific productivity (mass of product per mass of biocatalyst) by optimizing physiological (including genetic) and technological parameters. This sounds fairly straightforward, but experimental work has shown that neither the nutrient composition nor the biochemistry of the cell are as reproducible as a merely chemical process. Among food grade organisms, strains of Acetobacter are particularly well known for genetic instability. Subculturing is bound to generate mutants, some of which even may have lost the capability to oxidize ethanol.
Ralf G. Berger

### 12. Outlook

Abstract
The history of ‘bioflavors’ is long (cf. chapter 2, Sect. 5.1) and has, more recently, been successfully continued by bioprocesses for nonvolatile flavors, such as HFCS, MSG, or aspartame. As the above chapters have amply demonstrated, there have been numerous achievements in the field of volatile flavors, and there exist many more potential applications. One critical factor will be the formulation of achievable objectives and target compounds (cf. Sect. 11.3). A more rational development of both biocatalysts and processing techniques is now possible on the basis of accumulated knowledge.
Ralf G. Berger

### Backmatter

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