Figure 1. Overview on alkyl pyrazines. The most processed alkyl pyrazines1 -7 . Annual volumes of use of pyrazine derivatives used as flavoring agents in Europe 2004 is stated in kg/year, whereas the * represents the quantitative natural occurrence of alkyl pyrazines in foods in Europe in tonnes per year [6]. Special attention belongs to compounds 1 , 3 , 4, and 8 which are commercially available as pure substance declared as natural whereas others are at least partially chemical synthesized. Short notice: To the best of our knowledge these are the latest official volumes of the WHO and EFSA [7]. Non-commercial Synthetic alkyl-pyrazines11 -16 .
The consequences are high extraction costs and a product line that suffers from high risk of change in taste and smell. Therefore, industrial research is turning towards bio-based and/or biocatalytic strategies. Nowadays, the use of certain microorganisms [8] is well known and declared as natural and safe by food authorities [9] (EFSA, FDA etc. ), and offers the chance for a better economic feasibility with mild reaction conditions, high specificity and simplified downstream processes due to higher purities, better reproducibility and constant sourcing in contrast e.g. to the natural feedstock extraction.
Pyrazines are volatile, nitrogen-containing heterocyclic compounds widely distributed in plants [10], insects [11], fungi and bacteria [12]. They act as an odor signal to repel predators and effectively prevent vegetative tissue or immature fruit from being eaten [13]. For this reason, pyrazines find various applications as ingredients in pesticides, insecticides, dyes, and pharmaceutical compounds [14]. Pyrazines gained attention from the food industry as important ingredients in raw and roasted foods. Especially alkylated pyrazines are in the focus, as they have strong olfactory properties (Figure 1). They can be extracted from natural sources such as potatoes, nuts and coffee with applications in numerous areas, e.g. 2,3-dimethypyrazine 6 (coffee, nutty, roasty) is used in various beverages and sauces, while 2,5-dimethylpyrazine 3(nutty, musty, earthy, roasted cocoa) is used in breakfast cereals. 2,3,5-Trimethylpyrazine 1 (chocolate enhancer) and 2,6-dimethylpyrazine 4 (roasty, nutty, beef) are used in meals with coffee, cocoa, meat or potatoes as a roasted flavour. 2-Ethyl-3-methylpyrazine 2 (nutty, musty, corn, raw, earthy, oily) is used in peanut products, popcorn, and in bread. The perceived odor depends on the dilution (recommended 0.1% or less in dipropylene glycol) of the substance. Methoxylated pyrazines are mainly used in the perfume industry to improve the odor of cosmetics and toiletries [15] (fresh, green, woody, ambery, oriental, musky, minty and herbaceous). Pyrazine flavors are often added to microwave foods because roasting odors are not formed in microwaves at temperatures below 200°C [16].
The growing demand for the ‘natural‘ alkyl pyrazines described above cannot be satisfied by extraction and concentration techniques from natural sources alone, since the natural content of e.g. 0.01 wt. % pyrazines in sugar beet molasses is low [14c, 17]. Modern biotechnology, recognized as a sustainable economic process, could cope with the growing demand for ‘natural‘ compounds in the future and may further increase the amount may fulfil the market demand of selectively substituted pyrazines.