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Method and strain for producing tetramethylpyrazine

A technology of tetramethylpyrazine and bacterial strains, which is applied in the field of bioengineering, can solve problems such as failure to develop corresponding technologies, and achieve the effects of low production costs, mild conditions, and abundant sources of raw materials

Inactive Publication Date: 2011-01-26
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] On the basis of the previous research, our laboratory developed a strain of Bacillus subtilis XZ1124, which was fermented for 120 hours, and the yield of TTMP reached 3.92g / L; it was also found in the research that this strain can synthesize a large amount of TTMP precursor substances Acetoin, researchers speculate that acetoin may generate TTMP through biological enzyme catalysis (a strain of Bacillus subtilis with high tetramethylpyrazine production and its fermentation method for producing tetramethylpyrazine, patent application number: 200810235366.1) However, limited to the investigation of the characteristics of TTMP produced by microbial fermentation and the report on the regulation of fermentation based on the synthesis characteristics of TTMP, the corresponding technology has not been developed. Synthetic properties of TTMP to further increase the yield of TTMP

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  • Method and strain for producing tetramethylpyrazine
  • Method and strain for producing tetramethylpyrazine
  • Method and strain for producing tetramethylpyrazine

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Experimental program
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Embodiment 1

[0035] Embodiment 1: the verification that acetoin and ammonia react to generate TTMP

[0036] (1) Verification of extracellular enzymatic catalytic reaction: In order to verify whether there is an extracellular enzymatic catalytic reaction in the process of the reaction of precursor acetoin and ammonia to generate TTMP in the fermentation system, a model system is designed, which contains a certain concentration of Acetoin and 30g / L DAP adjusted the pH of the model system to 7.0.

[0037] Bacillus subtilis XZ1124 was inoculated in YG medium for shake flask culture. The fermented liquid cultured for 48 hours was taken, centrifuged at 8000 rpm for 15 minutes, the supernatant was passed through a 0.22 μm water membrane, and the obtained clarified liquid was used as the extracellular crude enzyme liquid. Add a certain volume of crude enzyme solution to the above-mentioned model system, and start the reaction after vigorous shaking, and take the reaction of adding the same volume...

Embodiment 2

[0047] Embodiment 2: Determination of TTMP synthesis pathway in microbial fermentation system

[0048] The process of Bacillus sp. to metabolize acetoin is generally believed to be the condensation of two molecules of pyruvate to form a molecule of α-acetolactate, and the decarboxylation of α-acetolactate under the action of α-acetolactate decarboxylase to form acetoin. Acetoin is reduced to 2,3-butanediol under the action of butanediol dehydrogenase, and the two can be transformed into each other.

[0049] Bacillus subtilis XZ1124 was inoculated on YG medium, cultured on a shaker at 37°C and 200r / min for 72 hours, the fermentation broth was extracted with ether, the extraction phase was qualitatively analyzed by GC-MS, and the spectra of each chromatographic peak were compared with NIST05a. The standard spectra in L Database (Agilent Technologies Inc.) were compared, and compared with the retention time of the standard, the following main metabolites were identified: acetoin,...

Embodiment 3

[0050] Example 3: Application of pH-controlled fermentation to improve the ability of microbial fermentation to accumulate precursor acetoin

[0051] Step 1: Activation of slant culture: Inoculate the B.licheniformis BL-L1 strain on broth agar medium, and culture it statically for 24-48 hours at 37°C, and set aside;

[0052] Step 2: Preparation of liquid seed culture: the bacterial strain cultivated in step 1, under aseptic conditions, pick 1-2 rings with an inoculation loop in a 250mL shaker flask equipped with 50mL liquid seed medium, and put it on a shaker at a rotating speed Cultivate at 200r / min and 37°C for 18-20h to obtain a liquid seed culture.

[0053] Step 3: Inoculate the liquid seed culture into a 7L fermenter with 5L of YG medium at an inoculum volume of 24%, set the stirring speed of the fermenter to 500rpm, the temperature to 35-40°C, and the ventilation rate to 1vvm , the fermentation time is 48h, the acid-base control fermented liquid pH value is 5.5-6.0 in t...

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Abstract

The invention discloses a method and a strain for producing tetramethylpyrazine (TTMP), in particular a two-step production process that a microorganism accumulates precursor acetoin by using the fermentation of reducing sugar and the acetoin and ammonia undergo a non-enzymatic reaction to synthesize the TTMP, and belongs to the technical field of bioengineering. The microorganism is any one of bacillus subtilis (CCTCC NO:M 208157), bacillus licheniformis (CGMCC NO:3961), bacillus licheniformis (CGMCC NO:3962) and bacillus licheniformis (CGMCC NO:3963). The strain obtains biomass and accumulates endogenous precursor acetoin by using the fermentation of the reducing sugar, and the acetoin and the ammonia in a fermentation system undergo the non-enzymatic reaction to synthesize the TTMP. The method has the advantages that: the obtained bacteria quantity is higher, the accumulation amount of the acetoin is effectively improved (38 to 44g / L); the high-concentration endogenous precursor acetoin and the ammonia can quickly react under proper conditions to form the TTMP (16 to 20g / L); and the use ratio (40.3 percent) of precursors is obviously improved due to the accumulation of endogenous acetoin and an in-situ fermentation environment.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for producing tetramethylpyrazine and a production strain thereof. Background technique [0002] Tetramethylpyrazine (TTMP), also known as ligustrazine, is a nitrogen-containing heterocyclic compound in which methyl groups are connected to the carbon atoms of the pyrazine ring. TTMP naturally exists in cocoa products, coffee, dairy products, meat, peanuts, hazelnuts, rum, whiskey and soybean products. As a natural flavor, it has aromas of roasting, peanuts, hazelnuts and cocoa. The deployment of cold drinks, meat, dairy products, cigarettes and other flavors; at the same time, TTMP, as the main active alkaloid component of the rhizome of Ligusticum wallichii, a traditional Chinese medicine for promoting blood circulation, promoting qi and removing blood stasis, has the functions of dilating blood vessels, improving microcirculation and inhibiting platelet aggregatio...

Claims

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Application Information

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IPC IPC(8): C12P17/12C12N1/20C12R1/125C12R1/10
Inventor 徐岩朱兵峰
Owner JIANGNAN UNIV
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