Pseudomonas perolens pyrazines

As for S-containing heterocycles, many N-containing heterocycles are also found in heat-treated foods as secondary flavours as a result of Maillard-type reactions between reducing sugars and amino acids. Pyrazines are N-heterocycles important contributors to the taste and aroma of roasted and toasted foods as well as vegetables and fermented foodstuffs. In cultures of Pseudomonas perolens ATCC 10757, amino acids such as valine, glycine and methionine were shown to... 

Scheme 23.22 Some nitrogen-containing flavour compounds produced by microorganisms, a Methylanthranilate formation from N-methyl methylanthranilate 1 Trametes sp., Polyporus sp. b Different pyrazines produced with microorganisms in optimised media 2 mutant strain from Pseudomonas perolens ATCC 10757 3 Bacillus subtilis, Brevibacterium linens 4 mutant strain of Corynebacterium glutamicum...

The influence of media components on the production of 2-methoxy-3-isopropyl and 2-methoxy-3-secbutyl pyrazines by Pseudomonas perolens (ATCC 10757) and selected mutants of this culture was studied. Pyrazine production was observed only during the stationary growth phase. The parent culture produced a maximum of 42 ng/mL pyrazines while selected mutants were found to produce a maximum of 15,760 ng/mL. The parent and mutant strains were found to exhibit similar responses to nutrient sources. Maximum pyrazine production was observed using pyruvate (1%), lactate (1%) or nutrient broth as carbon source. Nitrogen source had no influence on pyrazine production. Maximum pyrazine formation was observed when phosphate level ranged from 0.4 mM to 1.2 mM in the media. 

Because the potential for producing "natural" 2-methoxy-3-alkyl pyrazines via fermentation might be improved by optimizing media composition, we studied the influence of media components on the synthesis 2-methoxy-3-isopropyl pyrazine by cultures of Pseudomonas perolens and selected mutant strains. 

Effect of Carbon Source. The growth of Pseudomonas perolens as well as culture levels of 2-methoxy-3-isopropyl pyrazine in minimal salts plus 1% pyruvate are shown in Figure 1. High cell density was obtained but the presence of 2-methoxy-3-isopropyl pyrazine was not detected until after cell numbers were no longer increasing. At this point, the amount of pyrazine increased rapidly reaching a maximum on the fifth day. The growth and pyrazine synthesis with other carbon sources showed similar behavior. The results are presented in Table I, and plotted for selected carbon sources at 1%... 

Further investigation found that synthesis of tetramethyl pyrazine was not limited to the Pseudomonas perolens with the multiple auxotrophy. Any one of the high producing strains that was incubated with lactate or pyruvate at 2% concentration was capable of producing tetramethyl pyrazine. This compound appeared after the methoxy pyrazine levels had stopped increasing (Fig 3). This would suggest that a precursor for the methoxy pyrazines must first be depleted before the excess pyruvate would be directed to tetramethyl pyrazine synthesis. 

Although this work has shown how various media constituents and pathway manipulations can influence the synthesis of substituted pyrazines by Pseudomonas perolens, elucidation of the individual biosynthetic steps is needed to understand fully the controls and explore the possibility that other substituted pyrazines could be made. 

Alkyl methoxy pyrazines are an important class of aroma compounds exhibiting intense green bean/green pea aroma notes. Reineccius s group at the University of Minnesota show that mutant strains of Pseudomonas perolens produce the isopropyl isomer to a final level of 15 mg/L of culture. Since the threshold is 2xl0 ° ppm, the reported yield is substantial. 

As can be seen in Table 9.5, nutrient source had a strong influence on the production of the methoxy pyrazines by a selected mutant of Pseudomonas perolens. The end result is that an organism that initially produced a few ppb of the methoxy alkyl pyrazines was converted into an organism that produced ppm levels of the target compounds. While one may not think that ppm levels of an aroma compound is significant, these pyrazines have sensory thresholds in the pptr levels. 

Mclver, R.C., Factors Influencing the Synthesis of Methoxy Alkyl Pyrazines by Pseudomonas Perolens, Ph.D. thesis, University of Minnesota, St. Paul, p. 88, 1986. Kuila, R.K., B. Ranganathan, Ultraviolet light-induced mutants of Streptococcus lactis subspecies diacetylactis with enhanced acid- or flavor-producing ahihties, J. Dairy ScL, 61, 4, p. 379, 1978. 

Musty or potato-like flavor and aroma have been observed as a defect in milk (Hammer and Babel 1957) and Gruyere de Comte cheese (Dumont et al. 1975). This off-flavor results from the production of nitrogenous cyclic compounds by Pseudomonas taetrolens and P. perolens (Morgan 1976). Musty-flavored compounds produced by these organisms include 2,5-dimethylpyrazine and 2-methoxy-3-isopropyl-pyrazine. The Gruyere de Comte with potato off-flavor contained 3-methoxy-2-propyl pyridine, as well as alkyl pyrazine compounds (Dumont et al. 1975). Murray and Whitfield (1975) postulated that alkyl pyrazines are formed in vegetables by condensation of amino acids such as valine, isoleucine, and leucine with a 2-carbon compound. Details of the synthetic mechanism in pseudomonads are unknown. 

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