High-yield fermentative preparation of tetramethylpyrazine by Bacillus sp. using an endogenous precursor approach

Abstract

A spore-forming Bacillus sp. was isolated from a high-temperature Daqu, a starter culture of Chinese Maotai-flavor liquor, using an endogenous precursor screening strategy. The Bacillus sp. was capable of producing a high level of 2,3,5,6-tetramethylpyrazine (TTMP) via a precursor of 3-hydroxy-2-butanone (HB). The strain was characterized as Bacillus subtilis based on morphological, physiological, and biochemical properties as well as on partial 16S rRNA gene sequences. Different carbon and nitrogen sources as well as fermentation conditions were investigated. Optimization tests showed that oxygen supply and fermentation temperature were the most important parameters determining the production process. The production of >4.08 g/l TTMP was achieved together with a high level of endogenous precursor HB accumulation (>20 g/l) in both flask and fermentor cultures when the optimized medium and cultivation conditions were applied. Our data demonstrates the effectiveness of the endogenous precursor strategy for screening microorganisms that produce flavor compounds with structure-related precursors. The high yield of TTMP and the inexpensiveness of the agro-industrial product used as the substrate (soybean meal) indicate the potential of this process for industrial application.

Involvement of Colonizing Bacillus Isolates in Glucovanillin Hydrolysis during the Curing of Vanilla planifolia Andrews

“…Additionally, another B. subtilis isolate showed the ability to produce tetramethylpyrazine (a commonly occurring alkylpyrazine that is responsible for the odor of oriental foods, such as Chinese liquors) via the precursor 3-hydroxy-2-butanone (33). Combined with the findings of the present study, this suggests that Bacillus isolates can convert aromatic precursors and influence flavor formation (34,35).…”

A feeding strategy for tetramethylpyrazine production by Bacillus subtilis based on the stimulating effect of ammonium phosphate

“…One of them was centrifuged (10,0009g, 10 min) and the precipitate was collected, washed twice with distilled water and dried at 105°C to constant weight to determine the biomass of the culture according to Ji et al [12], the supernatant was used to determine total sugar using 3,5-dinitrosalicylate method as described by Miller [13]. Another aliquot was diluted to appropriate folds with phosphate buffer (0.5 M, pH 7.0), the dilution was then assayed for precursor HB and TTMP according to the literature [10].…”

“…strains [5][6][7]10], and kinds of methods were accordingly developed for enhanced TTMP production, such as enrichment of the medium with precursor 3-hydroxy-2-butanone (HB) [6,7], the employment of soytone and vitamin supplements [5] and accumulation of precursor HB endogenously from glucose [10]. However, little work has concentrated on the prominent effect of inorganic nitrogen source, such as ammonium salt on cell growth, precursor HB accumulation and corresponding TTMP production.…”

“…strain was isolated from a Chinese Maotai-flavor Daqu using an endogenous precursor strategy [10]. The newly isolated strain was capable of producing a high level of TTMP (4.08 g/l) with a high endogenous precursor HB production.…”

Quantification of volatile compounds in Chinese soy sauce aroma type liquor by stir bar sorptive extraction and gas chromatography–mass spectrometry

“…14 Recently, a high-yield strain of 2,3,5,6-tetramethylpyrazine was found in Chinese daqu, which showed that some pyrazines could be derived from the microbial products. 18 Six acids were identified in this work, the most abundant of which was butanoic acid, followed by hexanoic acid. These acids were produced by a diversity of microorganisms during fermentation of Chinese liquor.…”