CN101445786B - Bacillus subtilis highly producing tetramethylpyrazine and method thereof for fermentation producing tetramethylpyrazine - Google Patents
Bacillus subtilis highly producing tetramethylpyrazine and method thereof for fermentation producing tetramethylpyrazine Download PDFInfo
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Abstract
The invention relates to a bacillus subtilis highly producing tetramethylpyrazine and a method thereof for fermentation producing tetramethylpyrazine, and belongs to the technical field of bio-engineering. The invention discloses a Bacillus subtilis XZ1124 which can highly produce tetramethylpyrazine, and is preserved in CCTCC, and the preservation number is CCTCC NO: M 208157. The strain is madeof Maotai-flavor liquor and high temperature Daqu; the strain is identified as Bacillus subtilis based on colony, cell form, physiological and biochemical characteristics and 16S rRNA gene sequence comparative result thereof. The strain can utilizes glucose, sucrose, molasses and soybean cake powder as substrate, and increases the output of tetramethylpyrazine through accumulating a great deal ofendogenous precursors, so as to solve the problems of low product density, exogenous adding precursor requirement, and low precursor utilization rate in tetramethylpyrazine production through microbefermentation. When sucrose and soybean cake powder are used as substrate, 4.08 g/L of tetramethylpyrazine can obtained through shaking culture of the raw material for 120 h at the temperature of 37 DEG C.
Description
Technical field
The present invention relates to the subtilis that a plant height produces Tetramethylpyrazine, relate in particular to a bacillus subtilis and can accumulate the method that 3-hydroxyl-2-butanone is a precursor fermentative Production Tetramethylpyrazine in a large number, belong to technical field of bioengineering.
Background technology
Tetramethylpyrazine (2,3,5,6-tetramethylpyrazine) be a kind of common heterocyclic nitrogen, extensively be present in food raw material, processed food and the alcoholic beverage, be considered to the important perfume compound of a class, have the fragrance of baking, peanut, fibert and cocoa, be usually used in the allotment of essence such as bake, cold drink, meat, milk-product, cigarette.Also detect a certain amount of Tetramethylpyrazine and other alkyl pyrazine in the traditional liquor of China, they have lower taste threshold usually, therefore are considered to that the local flavor of Chinese Maotai-flavor liquor is had important contribution.Except purposes as the flavour of food products additive, Tetramethylpyrazine is as the main active alkaloid composition of Chinese medicinal materials Ligusticum wallichii (Ligusticum wallichii) rhizome, be proved to be pharmacological action, and oxidative stress, apoptosis and the renal toxicity of cisplatin induction had prophylactic effect with treatment cardiovascular and cerebrovascular diseases.Along with going deep into of pharmacological research, now be widely used in clinical as a kind of novel calcium ion antagonist, be large medication of circulation, nervus centralis, breathing and other system's relative disease, be widely used in the treatment of cardiovascular and cerebrovascular diseases, respiratory system disease, renal glomerular disease etc.
3-hydroxyl-2-butanone (3-hydroxy-2-butanone) is the meta-bolites that many microorganisms have physiological significance, and its precursor as Tetramethylpyrazine is applied to be confirmed by many investigators in the microbial fermentation production Tetramethylpyrazine.The method of adding precursor 3-hydroxyl-2-butanone by external source has been successfully applied in the output that improves the fermentative Production Tetramethylpyrazine, but there is certain technical difficulty in this technology, research level still rests on laboratory stage, does not carry out suitability for industrialized production as yet.
The chemical structure of Tetramethylpyrazine and 3-hydroxyl-2-butanone:
The production method of Tetramethylpyrazine can be divided into biological synthesis process, directly three kinds of extraction method and chemical synthesiss from plant.Up to now, the bibliographical information of existing a great deal of and patent utilization Maillard reaction and the synthetic pyrazine compounds of Strecker degraded chemical method, yet, the environmental issue that the chemical synthesis ubiquity is severe, reaction conditions is generally more violent, higher to equipment requirements, and the human consumer is more prone to use ' natural ' or ' biosynthesizing ' pyrazine, though the price of the pyrazine that chemosynthesis obtains is far below the former.It is limited that the direct method of extracting Tetramethylpyrazine from Chinese medicinal materials Ligusticum wallichii rhizome often faces the supply of Ligusticum wallichii material of vegetable origin, and the content of Tetramethylpyrazine in the Ligusticum wallichii rhizome lower (about 0.075%) causes the higher and very difficult realization scale operation of extraction process cost.Along with the day by day raising of people, utilize bionic means production Tetramethylpyrazine to have advantages such as Product Green is natural, cost is low, reaction conditions is gentle, environmental pollution is little and paid close attention to by the human consumer to the green product demand.
As far back as 1962, people such as Kosuge are separated to Tetramethylpyrazine first from the fermented liquid of Japanese Natto function capable of fermenting food bacterium Bacillus natto, in decades subsequently, multidigit investigator produces on the pyrazine field at microbe fermentation method and has made outstanding contribution, the bibliographical information of a great deal of in liquid state, produce the example of pyrazine in the solid-state and solid-state fermenting substrate, produce bacterial classification and comprise bacillus and mutant strain (Bacillus sp.) thereof, Corynebacterium glutamicum mutant (Corynebacterium glutamicum mutant) and lactobacillus (Lactococcuslactis subsp.Lactis biovar.Diacetylactis FC1) etc.According to above document, Bacillaceae (Bacillussp.) can utilize cheap industrial raw material to ferment as nutritive substance and generate Tetramethylpyrazine in a large number, is considered to the good pyrazine of a class and produces bacterial strain.
Though a lot of microorganisms can utilize the various high added value products of pathways metabolism ' de novo synthesis ' of self, these products content in the fermented liquid of microorganism generally very low (less than every liter of 0.60 gram), thus be difficult to carry out suitability for industrialized production.When the generation of tunning related to the precursor of similar, the method that adds precursor can increase substantially the effective ways of tunning and extensively used as a kind of.Equally, many documents has proved that in the substratum of microorganism the method for heavy addition 3-hydroxyl-2-butanone improves the feasibility of the output method of Tetramethylpyrazine.Yet this precursor that adds is utilized degree very low by microorganism, even under the situation that the higher amount pyrazine produces, and the utilization ratio of precursor also not high (less than 1%).Precursor 3-hydroxyl-2-butanone that a large amount of external sources are added adds in the substratum, cause the disorder of microbial metabolism equilibrated, various enzymes are suppressed by the high density precursor in the pathways metabolism of interior ' de novo synthesis ' precursor of microbe, though the concentration of product Tetramethylpyrazine obtains certain raising, but cell meets ' cell economics ' principle to the utilization of various nutritive substances in the environment, only there is a spot of precursor to participate in being rich in the pathways metabolism of microorganism under the precursor envrionment conditions, causes the utilization ratio of precursor lower.Simultaneously, the adding of a large amount of external source precursors and the generation of a certain amount of product have toxicity to microorganism cells usually, and the industrial application of bioprocess is had disadvantageous effect.In addition, the market demand of natural precursor 3-hydroxyl-2-butanone is still higher, therefore, does not meet economic feasibility by the method that adds precursor raising Tetramethylpyrazine output.In this case, screening can utilize glucose or other sugar efficiently to accumulate precursor 3-hydroxyl-2-butanone for carbon source material, thereby it is very necessary to reach the new bacterial strain of realizing the Tetramethylpyrazine high yield.
It is reported, can detect a series of pyrazine compounds in different types of Chinese traditional liquor, than the liquor of other type, the content of Tetramethylpyrazine is the highest in the Maotai-flavor liquor.The manufacture craft that high-temperature daqu is strict and complicated with it, with and as the saccharification and the starter of Maotai-flavor liquor manufacturing processed, be rich in various microorganisms and comprise bacterium, yeast and fungi.Bacillaceae (Bacillus sp.) can be survived under the low moisture high-temperature condition because of it becomes major microorganisms in the Daqu microorganism species system, the amylase of Bacillaceae (Bacillus sp.) microorganism secretion and proteolytic enzyme etc. make microorganism make full use of the nutritive substance in the environment, with sugar is that precursor 3-hydroxyl-2-butanone that nutrient metabolism produces also is greatly improved, therefore, the content of Tetramethylpyrazine correspondingly increases to some extent.As far as we know, most of alkyl pyrazine generation bacterium is the sporiferous Bacillaceae of a class.Simultaneously, derive from the wild Bacillaceae (Bacillus sp.) of food grade high-temperature daqu, its tunning can satisfy the demand of human consumer to natural product to a certain extent.
Summary of the invention
At the problem of prior art difficult point and existence, the object of the present invention is to provide the application of the new isolating subtilis of a strain in the fermentative Production Tetramethylpyrazine, this bacterial strain can improve the output of Tetramethylpyrazine with the method for source precursor in a large amount of accumulation.The pathways metabolism that bacterial strain of the present invention can utilize self transforming glucose or sucrose effectively generates the precursor 3-hydroxyl-2-butanone of Tetramethylpyrazine, under the biocatalysis of microbial enzyme, precursor transforms and generates Tetramethylpyrazine, thereby reaches the purpose of high yield Tetramethylpyrazine.
Technical scheme of the present invention: the bacterial strain of Tetramethylpyrazine is produced in a strain, and its classification called after subtilis (Bacillus subtilis) XZ1124 has been preserved in Chinese typical culture collection center, and its deposit number is CCTCC NO:M208157.
The bacterial characteristics of this subtilis is: the colonial morphology initial stage is grey, projection, circle, smooth surface is glossy, and the late stage of culture colony edge is irregular, flat, the canescence in surface, tarnish; Sediments microscope inspection is a rod-short, long 1.6-3.0 μ m, and diameter 0.6-0.7 μ m produces gemma; Physiological and biochemical property is Gram-positive, aerobic, chemoheterotrophic bacteria, produces acid, the catalase positive from glucose, fructose, seminose, maltose or sucrose, the nitrate reduction positive, the V-P reacting positive, hydrolyzable casein, gelatin and starch do not utilize Citrate trianion, propionic salt; The gene order of its 16S rRNA gene order and many bacillus subtilis (Bacillus subtilis) 16S rRNA has〉99% homology.
The above-mentioned substratum that is used for the thalli morphology observation is a broth agar culture medium, consists of (restraining every liter): glucose 5, peptone 10, extractum carnis 5, sodium-chlor 5, agar 15-20, pH7.0-7.2, distilled water preparation.
" the common bacteria system identification handbook " that the test method of above-mentioned colony morphology characteristic and physiological and biochemical test substratum and test method are write with reference to the elegant pearl in east, Cai Miaoying etc., Science Press, 2001, first version, p353-398.
The MR-VP substratum of the above-mentioned V-P of being used for test consists of (restraining every liter): glucose 5, peptone 5, sodium-chlor 5, pH7.2, distilled water preparation.
A kind of method that improves fermentation of bacillus subtilis method production Tetramethylpyrazine output: this subtilis is with dextrose culture-medium, sucrose medium, molasses culture medium, sucrose soybean cake powder substratum or molasses soybean cake powder substratum fermentative Production Tetramethylpyrazine, fermentation transforming glucose or sucrose and industrial raw material molasses and soybean cake powder generate the precursor 3-hydroxyl-2-butanone of Tetramethylpyrazine, under the biocatalysis of microbial enzyme, a large amount of precursor 3-hydroxyls that exist-2-butanone transforms and generates Tetramethylpyrazine.
The substratum of fermentative Production Tetramethylpyrazine is settled to 1L in g/L with distilled water, selects for use:
(1) dextrose culture-medium: glucose 100-150, peptone 30, yeast extract paste 10, Secondary ammonium phosphate 30, pH7.0-7.5;
(2) sucrose medium: sucrose 100-150, peptone 30, yeast extract paste 10, Secondary ammonium phosphate 30, pH7.0-7.5;
(3) sucrose soybean cake powder substratum: sucrose 100-150, soybean cake powder 40, yeast extract paste 5, Secondary ammonium phosphate 30, pH7.0-7.5;
(4) molasses culture medium: the molasses treatment solution accounts for the 21%-27% of culture volume, peptone 30, yeast extract paste 10, Secondary ammonium phosphate 30, pH7.0-7.5;
Or (5) molasses soybean cake powder substratum: the molasses treatment solution accounts for the 21%-27% of culture volume, soybean cake powder 40, yeast extract paste 5, Secondary ammonium phosphate 30, pH7.0-7.5.
Described subtilis CCTCC NO:M 208157, shake in the bottle at the 250mL that above-mentioned dextrose culture-medium of 50mL or sucrose medium are housed, under 37 ℃ of conditions, on shaking table with the rotating speed fermentation culture 120h of 200rpm, under the situation of a large amount of accumulation precursor 3-hydroxyl-2-butanone, obtaining concentration is the Tetramethylpyrazine of every liter of 3.25 or 3.68 gram.
Described subtilis CCTCC M 208157, shake in the bottle at the 250mL that the above-mentioned sucrose soybean cake powder of 50mL substratum is housed, under 37 ℃ of conditions, on shaking table with the rotating speed fermentation culture 120h of 200rpm, under the situation of a large amount of accumulation precursor 3-hydroxyl-2-butanone, obtaining concentration is the Tetramethylpyrazine of every liter of 4.08 gram.
Described subtilis CCTCC M 208157, shake in the bottle at the 250mL that the above-mentioned molasses culture medium of 50mL is housed, under 37 ℃ of conditions, on shaking table with the rotating speed fermentation culture 120h of 200rpm, under the situation of a large amount of accumulation precursor 3-hydroxyl-2-butanone, obtaining concentration is the Tetramethylpyrazine of every liter of 2.97 gram.
Described subtilis CCTCC M 208157, shake in the bottle at the 250mL that the above-mentioned molasses soybean cake powder of 50mL substratum is housed, under 37 ℃ of conditions, on shaking table with the rotating speed fermentation culture 120h of 200rpm, under the situation of a large amount of accumulation precursor 3-hydroxyl-2-butanone, obtaining concentration is the Tetramethylpyrazine of every liter of 3.19 gram.
Described subtilis CCTCC M 208157, in the 7L fermentor tank that the above-mentioned sucrose soybean cake powder of 4-5L substratum is housed, molasses treatment solution concentration is 24%, it is that 400-600rpm, temperature are 37 ℃, air flow 1vvm that mixing speed is set, the fermention medium initial pH value is 7.5, to add acid or alkali control fermented liquid pH value be 7.5 to stream in the fermenting process, fermentation time is 120 hours, under the situation of a large amount of accumulation precursor 3-hydroxyl-2-butanone, obtaining concentration is the Tetramethylpyrazine of every liter of 3.92 gram.
Beneficial effect of the present invention: the subtilis CCTCC NO:M208157 fermentative Production Tetramethylpyrazine of described high yield Tetramethylpyrazine can significantly improve the output of Tetramethylpyrazine by the endogenous precursor 3-of a large amount of accumulation hydroxyl-2-butanone.
Can ferment transforming glucose or sucrose and industrial raw material molasses and soybean cake powder of described subtilis CCTCC NO:M 208157 generates the precursor 3-hydroxyl-2-butanone of Tetramethylpyrazine, under the biocatalysis of microbial enzyme, a large amount of precursor 3-hydroxyls that exist-2-butanone transforms and generates Tetramethylpyrazine.
Described subtilis CCTCC NO:M 208157 derives from the high-temperature daqu of Maotai-flavor liquor, and its tunning Tetramethylpyrazine meets the relevant food safety code, to people, animal safety toxicological harmless, belongs to green safety food.
The concentration height of product Tetramethylpyrazine, the strain fermentation stable performance.
It is simple that this technology has production method, mild condition, and raw material sources enrich and characteristics such as production cost is low, have tempting prospects for commercial application.
The subtilis of high yield Tetramethylpyrazine provided by the invention, be Bacillus subtilis bacterial strain, solved tunning concentration is low in the Production by Microorganism Fermentation Tetramethylpyrazine, the need external source is added precursor raising Tetramethylpyrazine output and the low problem of external source interpolation precursor utilization ratio.Bacterium source of the present invention is in high-temperature daqu, tunning food safety height, can be the precursor 3-hydroxyl-2-butanone of the direct endogenous generation Tetramethylpyrazine of raw material with glucose or sucrose and industrial raw material molasses and soybean cake powder, thereby greatly promote the generation of Tetramethylpyrazine in the fermented liquid (the shake flask fermentation concentration of Tetramethylpyrazine can reach every liter of 4.08 gram).The technology that the method for microbe fermentation method by endogenous a large amount of accumulation precursors improves Tetramethylpyrazine output have that raw material is cheap and easy to get, reaction conditions is gentle, Product Green is natural and environmental friendliness etc. a bit.
The biological material specimens preservation
The bacterial strain of Tetramethylpyrazine is produced in one strain, its classification called after subtilis (Bacillus subtilis) XZ1124 has been preserved in Chinese typical culture collection center, is called for short CCTCC, preservation date on October 11st, 2008, its deposit number is CCTCC NO:M 208157.
Description of drawings
The gemma of Fig. 1 Bacillus subtilis of the present invention CCTCC NO:M 208157 bacterial strains amplifies 60000 times electromicroscopic photograph.
The analysis of Fig. 2 Bacillus subtilis of the present invention CCTCC NO:M 208157 bacterial strains fermented liquid in sucrose soybean cake powder substratum and the evaluation of Tetramethylpyrazine and precursor 3-hydroxyl-2-butanone.Identify through mass spectrum MS: the peak of 2.768min is interior mark 2-heptanone; 3.734min the peak be 3-hydroxyl-2-butanone, the peak of 6.067min is a Tetramethylpyrazine.
The situation of Fig. 3 Bacillus subtilis of the present invention CCTCC NO:M 208157 bacterial strains fermentation producing tetramethylpyrazine in sucrose soybean cake powder substratum.
Embodiment
Embodiment 1: the screening of the subtilis Bacillus subtilis CCTCC NO:M208157 bacterial strain of high yield Tetramethylpyrazine
The bacterium source that the present invention relates to is in the high-temperature daqu of producing ' openning cellar for storing things wine ', ' Lang Jiu ', ' edge this life ' and Maotai-flavor liquors such as ' Maotai '.Get a certain amount of above-mentioned Daqu, pulverize the back and add and fill in an amount of stroke-physiological saline solution of granulated glass sphere, vibration is 24 hours on 37 ℃, 150rpm shaking table; The bacteria suspension of making is in 80 ℃ of water-bath 20min, and flowing water cools off; Get an amount of above-mentioned bacteria suspension aseptic technique and insert in the broth culture, 37 ℃, 150rpm are cultivated 18h, and the gradient dilution spread plate is in 37 ℃ of cultivation 48h; Picking growth better, the different colony inoculations of proterties are in the bouillon agar inclined-plane, as the screening bacterial strain.
In above-mentioned bacterium inoculation MR-VP substratum, cultivate 48h for 37 ℃, carry out the V-P test by the Meara method, getting the 1ml fermented liquid adds 1ml and contains 40% NaOH solution of 0.3% creatine in test tube, concussion 1min, be statically placed in 30 ℃ of incubator 10min, Yihong look occur with mixed solution and represent the V-P test positive, the darker above-mentioned bacterial strains that gets colors is as the primary dcreening operation bacterial strain.
Above-mentioned bacterial strains is inoculated in the PYG substratum of modification, cultivate 48h for 37 ℃, get the concentration that a certain amount of fermented liquid GC analyzes Tetramethylpyrazine and 3-hydroxyl-2-butanone, choose the highest bacterial strain of Tetramethylpyrazine output, through the separating for several times screening, obtain the XZ-1124 bacterial strain again.
Show by the Physiology and biochemistry qualification test, the bacterial strain that above-mentioned test obtains is Gram-positive, aerobic, chemoheterotrophic bacteria, produce acid from glucose, fructose, seminose, maltose, sucrose, the catalase positive, the nitrate reduction positive, the V-P reacting positive, hydrolyzable casein, gelatin and starch do not utilize Citrate trianion, propionic salt; The gene order of its 16S rRNA gene order and many bacillus subtilis (Bacillus subtilis) 16S rRNA has high homology (〉 99%).
The preparation of the liquid seeds culture of embodiment 2:CCTCC NO:M 208157 bacterial strains
Step 1: slant culture activation: CCTCC NO:M 208157 bacterial classification inoculations in broth agar culture medium, under 37 ℃ of conditions, are left standstill and cultivate 24-48h, standby;
Step 2: the preparation of liquid seeds culture: with the bacterial strain of step 1 cultivation, choosing 1-2 with transfering loop under aseptic condition encircles in the 250ml that the 30-50ml liquid seed culture medium is housed and shakes in the bottle, put on the shaking table and to be 150-200rpm, 35-40 ℃ with the rotation rotating speed and to cultivate 16-20 hour, promptly make the liquid seeds culture.
Embodiment 3: utilize above-mentioned CCTCC NO:M 208157 bacterial strains fermentative Production Tetramethylpyrazine in sucrose medium.
Is that 5% inoculum size is inoculated into the 250mL that sterilized 50mL sucrose medium is housed and shakes in the bottle with the liquid seeds culture of CCTCC NO:M 208157 bacterial strains of the foregoing description 2 with volume percent, in rotating speed is on the shaking table of 200rpm, cultivated 96-120 hour for 37 ℃, the accumulation volume of precursor 3-hydroxyl-2-butanone is every liter of 31.0 gram in the fermented liquid, and the concentration of Tetramethylpyrazine can reach every liter of 3.68 gram.
The composition of above-mentioned sucrose medium is (gram, every liter of distilled water): sucrose 100, and peptone 30, yeast extract paste 10, Secondary ammonium phosphate 30 is regulated the initial pH7.5 of substratum.
Embodiment 4: utilize above-mentioned CCTCC NO:M 208157 bacterial strains fermentative Production Tetramethylpyrazine in dextrose culture-medium.
With the liquid seeds culture of CCTCC NO:M 208157 bacterial strains of the foregoing description 2 is that the inoculum size of 2-5% is inoculated into the 250ml that sterilized 50ml dextrose culture-medium is housed and shakes in the bottle with the volume percent, in rotating speed is on the shaking table of 200rpm, cultivated 96-120 hour for 37 ℃, the accumulation volume of precursor 3-hydroxyl-2-butanone is every liter of 28.2 gram in the fermented liquid, and the concentration of Tetramethylpyrazine can reach every liter of 3.25 gram.
The composition of above-mentioned dextrose culture-medium is (gram, every liter of distilled water): glucose 100, and peptone 30, yeast extract paste 10, Secondary ammonium phosphate 30 is regulated the initial pH7.5 of substratum.
Embodiment 5: utilize above-mentioned CCTCC NO:M 208157 bacterial strains fermentative Production Tetramethylpyrazine in molasses culture medium.
With the liquid seeds culture of CCTCC NO:M 208157 bacterial strains of the foregoing description 2 is that the inoculum size of 2%-5% is inoculated into the 250mL that sterilized 50mL sucrose soybean cake powder substratum is housed and shakes in the bottle with the volume percent, in rotating speed is on the shaking table of 200rpm, cultivated 96-120 hour for 37 ℃, the accumulation volume of precursor 3-hydroxyl-2-butanone is every liter of 26.6 gram in the fermented liquid, and the concentration of Tetramethylpyrazine can reach every liter of 2.97 gram.
The composition of above-mentioned molasses culture medium is (gram, every liter of distilled water): molasses treatment solution 24% (v/v), and peptone 30, yeast extract paste 10, Secondary ammonium phosphate 30 is regulated the initial pH7.5 of substratum.
Embodiment 6: utilize above-mentioned CCTCC NO:M 208157 bacterial strains fermentative Production Tetramethylpyrazine in sucrose soybean cake powder substratum.
With the liquid seeds culture of CCTCC NO:M 208157 bacterial strains of the foregoing description 2 is that the inoculum size of 2-5% is inoculated into the 250mL that sterilized 50mL sucrose soybean cake powder substratum is housed and shakes in the bottle with the volume percent, in rotating speed is on the shaking table of 200rpm, cultivated 96-120 hour for 37 ℃, the accumulation volume of precursor 3-hydroxyl-2-butanone is every liter of 34.7 gram in the fermented liquid, and the concentration of Tetramethylpyrazine can reach every liter of 4.08 gram.
The composition of above-mentioned sucrose soybean cake powder substratum is (gram, every liter of distilled water): sucrose 100, and soybean cake powder 40, yeast extract paste 5, Secondary ammonium phosphate 30 is regulated the initial pH7.5 of substratum.
Embodiment 7: utilize above-mentioned CCTCC NO:M 208157 bacterial strains fermentative Production Tetramethylpyrazine in molasses soybean cake powder substratum.
With the liquid seeds culture of CCTCC NO:M 208157 bacterial strains of the foregoing description 2 is that the inoculum size of 2-5% is inoculated into the 250mL that sterilized 50mL sucrose soybean cake powder substratum is housed and shakes in the bottle with the volume percent, in rotating speed is on the shaking table of 200rpm, cultivated 96-120 hour for 37 ℃, the accumulation volume of precursor 3-hydroxyl-2-butanone is every liter of 29.5 gram in the fermented liquid, and the concentration of Tetramethylpyrazine can reach every liter of 3.19 gram.
The composition of above-mentioned molasses soybean cake powder substratum is (gram, every liter of distilled water): molasses treatment solution 24% (v/v), and soybean cake powder 40, yeast extract paste 5, Secondary ammonium phosphate 30 is regulated the initial pH7.5 of substratum.
Embodiment 8: utilize above-mentioned CCTCC NO:M 208157 bacterial strains in 7L fermentation cylinder for fermentation method production Tetramethylpyrazine.
Is inoculum size with the volume ratio of 2%-5% with the liquid seeds culture of CCTCC NO:M 208157 bacterial strains of the foregoing description 2 with volume percent, be inoculated in the 7L fermentor tank that 4-5L sucrose soybean cake powder fermention medium is housed, it is 400-600rpm that the fermentor tank mixing speed is set, temperature is 35-40 ℃, air flow 1vvm, fermentation time is 96-112 hour, the fermention medium initial pH value is 7.0-7.5, to add soda acid control fermented liquid pH value be 7.0 to stream in the fermenting process, glucose in the fermenting process in the interval certain hour sampling analysis fermented liquid, the content of Tetramethylpyrazine and 3-hydroxyl-2-butanone finishes fermentation when the concentration of Tetramethylpyrazine in the fermented liquid no longer rises.Fermented 120 hours, the accumulation volume of precursor 3-hydroxyl-2-butanone is every liter of 33.7 gram in the fermented liquid, and the concentration of Tetramethylpyrazine can reach every liter of 3.92 gram.
The composition of above-mentioned sucrose soybean cake powder substratum is (gram, every liter of distilled water): sucrose 100, and soybean cake powder 40, yeast extract paste 5, Secondary ammonium phosphate 30 is regulated the initial pH7.5 of substratum.
Utilize that the fermented liquid component analyzing method is in the above-mentioned Bacillus subtilis CCTCC NO:M 208157 strain fermentation method production Tetramethylpyrazine processes: get above-mentioned fermented liquid with the centrifugal 8-10 of 8000-10000rpm minute, measure the content of glucose in the supernatant liquor, get the 5mL supernatant liquor and add a certain amount of dichloromethane extraction, with the content of Tetramethylpyrazine and 3-hydroxyl-2-butanone in the gas phase mensuration extraction phase.
The method of above-mentioned chromatogram ration analysis is: GC6890, use the DB-Wax post, and carrier gas is nitrogen (N
2); 250 ℃ of injector temperatures, 250 ℃ of detector temperatures; Temperature programming: kept 2 minutes for 50 ℃, the speed with 10 ℃/min is raised to 210 ℃ then, keeps 2 minutes; Sample size 1 μ L; Splitting ratio is 2.The retention time of 3-hydroxyl-2-butanone is 3.7 minutes under these conditions, and the retention time of Tetramethylpyrazine is 6.1 minutes.
Claims (4)
1. the bacterial strain of Tetramethylpyrazine is produced in a strain, and its called after subtilis (Bacillus subtilis) XZ1124 has been preserved in Chinese typical culture collection center, and its deposit number is CCTCC NO:M208157.
2. bacterial strain according to claim 1, the bacterial characteristics that it is characterized in that this subtilis is: the colonial morphology initial stage is grey, projection, circle, smooth surface is glossy, the late stage of culture colony edge is irregular, flat, the canescence in surface, tarnish; Sediments microscope inspection is a rod-short, long 1.6-3.0 μ m, and diameter 0.6-0.7 μ m produces gemma; Physiological and biochemical property is Gram-positive, aerobic, chemoheterotrophic bacteria, produces acid, the catalase positive from glucose, fructose, seminose, maltose or sucrose, the nitrate reduction positive, the V-P reacting positive, hydrolyzable casein, gelatin and starch do not utilize Citrate trianion, propionic salt; The gene order of its 16S rRNA gene order and many bacillus subtilis (Bacillus subtilis) 16S rRNA has＞99% homology.
3. in the method for fermentation of bacillus subtilis production Tetramethylpyrazine, improve the method for Tetramethylpyrazine output, it is characterized in that subtilis CCTCC NO:M 208157 shakes in the bottle at the 250mL that 50mL dextrose culture-medium or sucrose medium are housed, under 37 ℃ of conditions, on shaking table with the rotating speed fermentation culture 120h of 200rpm, under the situation of a large amount of accumulation precursor 3-hydroxyl-2-butanone, obtaining concentration is the Tetramethylpyrazine of every liter of 3.25 or 3.68 gram;
Described dextrose culture-medium is settled to 1L with distilled water: glucose 100-150g/L, peptone 30g/L, yeast extract paste 10g/L, Secondary ammonium phosphate 30g/L, pH7.0-7.5;
Described sucrose medium is settled to 1L with distilled water: sucrose 100-150g/L, peptone 30g/L, yeast extract paste 10g/L, Secondary ammonium phosphate 30g/L, pH7.0-7.5.
4. in the method for fermentation of bacillus subtilis production Tetramethylpyrazine, improve the method for Tetramethylpyrazine output, it is characterized in that subtilis CCTCC M 208157 shakes in the bottle at the 250mL that 50mL sucrose soybean cake powder substratum is housed, under 37 ℃ of conditions, on shaking table with the rotating speed fermentation culture 120h of 200rpm, under the situation of a large amount of accumulation precursor 3-hydroxyl-2-butanone, obtaining concentration is the Tetramethylpyrazine of every liter of 4.08 gram;
Described sucrose soybean cake powder substratum is settled to 1L with distilled water: sucrose 100-150g/L, soybean cake powder 40g/L, yeast extract paste 5g/L, Secondary ammonium phosphate 30g/L, pH7.0-7.5.
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| CN101906448B (en) * | 2010-02-02 | 2012-07-11 | 浙江大学 | Method for biosynthesizing ligustrazine |
| CN101955980B (en) * | 2010-07-28 | 2013-05-22 | 江南大学 | Method and strain for producing tetramethylpyrazine |
| CN102618474B (en) * | 2012-04-10 | 2013-08-14 | 江苏今世缘酒业股份有限公司 | Bacillus subtilis and separate culture method for same |
| CN102994317B (en) * | 2012-12-27 | 2014-06-04 | 山东景芝酒业股份有限公司 | Production method of strong aromatic Chinese spirits |
| CN103865833A (en) * | 2013-10-25 | 2014-06-18 | 中国食品发酵工业研究院 | Preparation method of thermophilic bacillus subtilis microbial inoculum |
| CN106987546A (en) * | 2017-05-24 | 2017-07-28 | 安徽宣酒集团股份有限公司 | Tetramethylpyrazine bacillus amyloliquefaciens and the special wheat bran purposes of distilled spirit with sesame flavour |
| CN107475159B (en) * | 2017-09-19 | 2019-10-25 | 湖北白云边酒业股份有限公司 | Bacillus subtilis and its application in Sauce flavor white wine |
| CN110172435B (en) * | 2019-06-06 | 2020-12-29 | 江南大学 | Recombinant bacterium for catalytic synthesis of 2, 5-dimethylpyrazine |
| CN110305913B (en) * | 2019-06-28 | 2022-04-12 | 山东省食品发酵工业研究设计院 | Method for producing tetramethylpyrazine from sugar raw material |
| CN112662597B (en) * | 2021-01-26 | 2021-10-01 | 庐山市绿游生态农业开发有限公司 | Bacillus pumilus for high yield of trimethyl pyrazine and tetramethyl pyrazine |
| CN113583902B (en) * | 2021-07-23 | 2023-06-09 | 四川省宜宾市叙府酒业股份有限公司 | Strain for high yield of tetramethylpyrazine and preparation method thereof |
| CN114854617A (en) * | 2022-03-01 | 2022-08-05 | 四川大学 | Bacillus amyloliquefaciens for high-yield tetramethylpyrazine and solid state fermentation simulation method thereof |
| CN115386525B (en) * | 2022-10-26 | 2023-01-31 | 中粮营养健康研究院有限公司 | Bacillus subtilis, microbial inoculum, application and method for preparing tetramethylpyrazine |
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