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Bacillus megaterium, isolated in a screening process from topsoil, was used for N-demethylation of natural N-methyl methyl anthranilate to produce natural methyl anthranilate. Maximal productivity of 70 mg/L/day was achieved under laboratory-scale conditions without further optimization. No byproducts were observed. Thus, production of “natural” methyl anthranilate using B. megaterium is a significant improvement over comparable already existing procedures. Keywords: Bacillus megaterium; bioflavor; biotransformation; methyl anthranilate; N-demethylation; N-methyl methyl anthranilate

For the authenticity assessment of (E)-methyl cinnamate from different origins, combustion/pyrolysis-isotope ratio mass spectrometry (C/P-IRMS) was used by an elemental analyzer (EA) and on-line capillary gas chromatography coupling (HRGC-C/P-IRMS). For that reason, (E)-methyl cinnamate self-prepared from synthetic, natural, and semisynthetic educts was analyzed in comparison to the commercial synthetic and natural ester. In addition, (E)-methyl cinnamate from basil extract and a number of commercial natural aromas was investigated. The data of self-synthesized synthetic (E)-methyl cinnamate, i.e., delta(13)C(V)(-)(PDB) = -33.8 per thousand and delta(2)H(V)(-)(SMOW) = +349 per thousand, corresponded with that found for the commercial synthetic samples (-29.5 to -31.4 per thousand and +328 to +360 per thousand for delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW), respectively). The ester produced from natural educts by acid as well as Candida antarctica catalysis revealed delta(13)C(V)(-)(PDB) = -25.6 and -30.1 per thousand as well as delta(2)H(V)(-)(SMOW) = -162 and -169 per thousand, respectively. Acid-catalyzed semisynthetic products differed in their delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW) values depending on the origin of their educts. For the ester from synthetic methanol and natural cinnamic acid, -27.3 and -126 per thousand were determined for delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW), respectively, whereas for the ester produced from natural methanol and synthetic acid delta(13)C(V)(-)(PDB) = -30.6 per thousand and delta(2)H(V)(-)(SMOW) = +287 per thousand were found. Basil extract showed -28.9 and -133 per thousand for delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW), respectively. Commercial aromas declared to be natural revealed delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW) data ranging from -25.7 to -28.5 per thousand as well as -85 to -191 per thousand, respectively, indicating, in part, incorrect declaration.

By use of extracts prepared by liquid-liquid separation of the volatiles from self-prepared juices of pineapple fruits (Ananas comosus) (n = 14) as well as commercial pineapple recovery aromas/water phases (n = 3), on-line capillary gas chromatography-isotope ratio mass spectrometry was employed in the combustion (C) and the pyrolysis (P) modes (HRGC-C/P-IRMS) to determine the delta(13)C(VPDB) and delta(2)H(VSMOW) values of selected pineapple flavor constituents. In addition to methyl 2-methylbutanoate 1, ethyl 2-methylbutanoate 2, methyl hexanoate 3, ethyl hexanoate 4, and 2,5-dimethyl-4-methoxy-3[2H]-furanone 5, each originating from the fruit, the delta(13)C(VPDB) and delta(2)H(VSMOW) data of commercial synthetic 1-5 and "natural" (biotechnologically derived) 1-4 were determined. With delta(13)C(VPDB) data of pineapple volatiles 1-4 varying from -12.8 to -24.4 per thousand, the range expected for CAM metabolism was observed. Compound 5 showed higher depletion from -20.9 to -28.6 per thousand. A similar situation was given for the delta(2)H(VSMOW) values of 3-5 from pineapple ranging from -118 to -191 per thousand, whereas 1 and 2 showed higher depleted values from -184 to -263 per thousand. In nearly all cases, analytical differentiation of 1-5 from pineapple and natural as well as synthetic origin was possible. In general, natural and synthetic 1-5 exhibited delta(13)C(VPDB) data ranging from -11.8 to -32.2 per thousand and -22.7 to -35.9 per thousand, respectively. Their delta(2)H(VSMOW) data were in the range from -242 to -323 per thousand and -49 to -163 per thousand, respectively.

By use of headspace SPME sampling and a PLOT column, on-line capillary gas chromatography-isotope ratio mass spectrometry was employed in the combustion (C) and the pyrolysis (P) modes (HRGC-C/P-IRMS) to determine the delta(13)C(VPDB) and delta(18)O(VSMOW) values of ethanol in authentic (n = 14) and commercial tequila samples (n = 15) as well as a number of other spirits (n = 23). Whereas with delta(13)C(VPDB) values ranging from -12.1 to -13.2 per thousand and from -12.5 to -14.8 per thousand similar variations were found for 100% agave and mixed tequilas, respectively, the delta(18)O(VSMOW) data differed slightly within these categories: ranges from +22.1 to +22.8 per thousand and +20.8 to +21.7 per thousand were determined for both the authentic 100% agave and mixed products, respectively. The data recorded for commercial tequilas were less homogeneous; delta(13)C(VPDB) data from -10.6 to -13.9 per thousand and delta(18)O(VSMOW) values from +15.5 to +22.7 per thousand were determined in tequilas of both categories. Owing to overlapping data, attempts to differentiate between white, rested, and aged tequilas within each of the two categories failed. In addition, discrimination of tequila samples from other spirits by means of delta(13)C(VPDB) and delta(18)O(VSMOW) data of ethanol was restricted to the products originating from C(3) as well as C(4)/CAM raw materials.