CN101705261A - Preparation method of Gamma-aminobutyric acid - Google Patents
Preparation method of Gamma-aminobutyric acid Download PDFInfo
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- CN101705261A CN101705261A CN 200910235693 CN200910235693A CN101705261A CN 101705261 A CN101705261 A CN 101705261A CN 200910235693 CN200910235693 CN 200910235693 CN 200910235693 A CN200910235693 A CN 200910235693A CN 101705261 A CN101705261 A CN 101705261A
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Abstract
The invention relates to a preparation method of Gamma-aminobutyric acid. The method includes the following steps: inoculating cultured Gamma-aminobutyric acid production bacterial strain to a fermentation culture medium in a fermentation tank for culturing, conducting combined fermentation of an aerobic section and an anaerobic section, controlling temperature in sections during the fermentation process and adopting a means of regulating in sections based on the demand for oxygen so that the Gamma-aminobutyric acid production bacterial strain is metabolized under an anaerobic state to produce Gamma-aminobutyric acid. The preparation method adopts ventilation in sections and high temperature fermentation, shortens the fermentation cycle, reduces the consumption of cooling water and cost and improves the yield of the Gamma-aminobutyric acid. The production process is simple, safe and energy-saving. The hydrolysis liquid of corn cob is used as fermentation raw material, thus not only changing wastes into valuables and reducing environment pollution, but also having positive significance in guaranteeing food security.
Description
Technical field
The invention belongs to the biological fermentation field, the present invention relates to a kind of preparation method of γ-An Jidingsuan.
Background technology
(4-Aminobutanoica cid, 4-AB), gamma aminobutyric acid, molecular formula are C to γ-An Jidingsuan (γ-Aminobutanoic acid is called for short GABA) to have another name called the 4-aminobutyric acid
4H
9NO
2, relative molecular weight 103.2 is a kind of white or near-white crystalline powder, and is very easily water-soluble, be slightly soluble in hot ethanol, be insoluble to cold ethanol, benzene, ether, fusing point (mp): 203-204 ℃ (decomposition), fusing is resolved into and is adjoined pyrrolidone and water, the dissociation constant of GABA: pKCOOH=4.03, pKNH
3=10.56, no opticity.
GABA a kind ofly extensively is present in prokaryotic organism and Eukaryotic nonprotein amino acid with free state.Studies show that of nervous physiology and neuromedicine, GABA is the inhibitory transmitter substance of central nervous system, it is one of most important neurotransmitter in the cerebral tissue, be the active amino acid that plays an important role in the human brain energy metabolism, it has glucose metabolism in the brain of activation, and short vagusstoff is synthetic, blood ammonia falls, anticonvulsion, hypotensive, the function of recovery brain cell.Up-to-date studies show that, GABA also has ataraxy effect, kidney function and liver function improvement effect, promote the alcohol metabolism effect and disappear smelly and effect such as high-efficient fat reducing.GABA both can develop becomes a kind of medicine with remarkable pharmacological action, and can develop again becomes a kind of food with health, and prospect is very optimistic.
GABA can be formed by the L-glutamic acid conversion under the stronger L-Glutamic decarboxylase effect of specificity, the difficulty but the accumulation meeting of GABA becomes with advancing age, and diet can effectively improve this situation, to help the health of human body.
Because the natural amount of GABA is low, be difficult to a large amount of extraction separation from some natural tissues, the method that obtains GABA at present is chemical synthesis and biological process two big classes, wherein biological process comprises in the plant two kinds of enrichments and microbe fermentation method.
The chemosynthesis of GABA has over one hundred year history, mainly is (180 ℃) reaction under strong condition with O-phthalic imide potassium and γ-neoprene cyanogen, and product refluxes with the vitriol oil, gets through crystallization and purification again.Another kind method is to make through calcium hydroxide, bicarbonate of ammonia hydrolysis by pyrrolidone.Generally speaking, chemical synthesis prepares GABA owing to used the stronger solvent of some corrodibility in production technique, reaction conditions is violent, and reason such as yield is lower, it is difficult in the food processing and production is used, products obtained therefrom can not be considered to a kind of natural foodstuff additive, has certain potential safety hazard.
The GABA enrichment is relevant with the stress reaction under its residing adverse environmental factor in the plant, and these stress reactions comprise hypoxemia, low temperature, arid, high H
+Concentration, physical abuse, dark and insect pest etc.Under the adverse environmental factor, Ca in the cell
2+Concentration increases fast, causes the accumulation of GABA, and the GABA of accumulation Ca in the irritation cell conversely
2+Continuation discharge, thereby strengthened the reaction of plant materials to adverse circumstance.
Fermentation method is to be conversion of substrate with the L-Sodium Glutamate, adds carbon source, nitrogenous source and inorganic salt and forms fermention medium, utilizes γ-An Jidingsuan to produce bacterial strain and transforms the preparation γ-An Jidingsuan.Comparatively speaking, the chemosynthesis security is relatively poor, and plants enriched GABA content is very low, and microbial fermentation synthetic GABA just seems to have DEVELOPMENT PROSPECT.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of γ-An Jidingsuan to improve the productive rate of γ-An Jidingsuan, reduces production costs.
The invention provides a kind of preparation method of γ-An Jidingsuan, it comprises the steps:
Will be in fermention medium through the γ-An Jidingsuan production inoculation of cultivating, carry out aerobic, two sections combined fermentations of anaerobism, fs is the aerobic fermentation stage, control pH5.5～6.0, keeping oxyty is 60～90%, temperature is controlled at 30～32 ℃, aerobic cultivation 10～20 hours, when cell concn is that optical density(OD) (O.D.) is when reaching 15～30, change the subordinate phase anaerobically fermenting over to, anaerobism is cultivated until fermentation ends, and the anaerobic stages temperature is controlled at 35～38 ℃, fermented 40～50 hours, and promptly obtained γ-An Jidingsuan.
It is newborn tyrothricin CGMCC NO.1306 (Lactobacillus brevis CGMCC NO.1306) that described γ-An Jidingsuan is produced bacterial strain.
Carbon source is one or more and the L-Sodium Glutamate in corn cob hydrolyzed solution, straw hydrolyzed solution or the bagasse hydrolysis sugar in the described fermention medium.
Nitrogenous source is a yeast extract paste, peptone, corn steep liquor, urea, ammonium sulfate, one or more in wheat bran hydrolyzed solution or the soybean meal hydrolysate.
Fermention medium also needs to add other mineral ion composition: MgSO of following weight per-cent except that adding carbon source and nitrogenous source
47H
2O 0.001～0.003%, MnSO
44H
2O0.004～0.006%, KH
2PO
40.02～0.07%, Soduxin 0.5～0.7%.
Preferably, fermentation tank culture medium comprises the component of following weight per-cent: corn cob hydrolyzed solution 20～30%, yeast extract paste 0.2～0.4%, peptone 0.1～0.3%, corn steep liquor 0.4～0.6%, MgSO
47H
2O 0.001～0.003%, MnSO
44H
2O 0.004～0.006%, KH
2PO
40.02～0.07%, Soduxin 0.5～0.7%, L-Sodium Glutamate 5～7%, all the other are water.
Wherein, the preparation process of corn cob hydrolyzed solution of the present invention is:
1) corncob acid hydrolysis: with corn cob in the sulphuric acid soln of concentration expressed in percentage by volume 0.1～10%, be hydrolyzed acid hydrolysis solution, hydrolysis temperature is 40～250 ℃, hydrolysis time 5～600 minutes;
2) solid-liquid separation: the acid hydrolysis solution solid-liquid separation, first liquid glucose and the corncob acid hydrolysis slag of wood sugar is rich in acquisition;
3) acidolysis slag enzymolysis: one or more in usefulness cellulase, zytase or the cellobiase, the corncob acid hydrolysis slag is carried out enzymic hydrolysis, get enzymolysis solution; Enzyme dosage is: every gram acidolysis slag 2～100PFIU cellulase, 1～50IU cellobiase, 10～500PFIU zytase; Control pH is 2.0～7.0 in the enzymolysis process, and temperature is 20～80 ℃, and enzymolysis time is 10～240 hours;
4) solid-liquid separation: the enzymolysis slag that the enzymolysis solution solid-liquid separation obtains being rich in second liquid glucose of glucose and is rich in xylogen.
Second liquid glucose is corn cob hydrolyzed solution of the present invention, wherein contains 6～8% glucose and 2～3% wood sugar and a small amount of pectinose.
The present invention is in fs aerobic fermentation process, by being 1 with sterile air with ventilating ratio: the rate of venting of (0.8～1.2) flows in the fermention medium, and mixing speed is 300～500rpm, control oxyty 60～90%.
The agent of described fermention medium pH regulator is selected from one or more in ammoniacal liquor, sodium hydroxide, yellow soda ash, lime carbonate, the calcium hydroxide.
Through aerobic, two stages of anaerobism leavening temperature is risen to 38 ℃ from 30 ℃ in the fermenting process of the present invention.
Specifically, a kind of corn cob hydrolyzed solution of the present invention prepares the method for γ-An Jidingsuan, and it comprises the steps:
1) the dull and stereotyped cultivation: adopting the corn cob hydrolyzed solution to add nitrogenous source is plate culture medium, γ-An Jidingsuan is produced bacterial strain receive in the plate culture medium that contains the corn cob hydrolyzed solution, cultivates 18～26 hours for 30 ℃;
2) shake-flask seed is cultivated: the γ-An Jidingsuan production bacterial strain that flat board is cultivated is received in the shake-flask seed substratum that contains the corn cob hydrolyzed solution, cultivated 20～24 hours for 30～33 ℃, pH is controlled at 5.0～5.5, shaking speed 180～220rpm;
3) seeding tank seed culture: the γ-An Jidingsuan of shake-flask seed culture medium culturing is produced bacterial strain receive in the seed tank culture base that contains the corn cob hydrolyzed solution, inoculum size is 5～10% (v/v), cultivated 15～20 hours for 30～33 ℃, pH is controlled at 5.0～5.5, mixing speed 75～100rpm, ventilating ratio 1: (0.6～0.8);
4) fermentor cultivation: the γ-An Jidingsuan that the seed tank culture base is cultivated is produced bacterial strain and is received in the 5L fermentor tank that contains the corn cob hydrolyzed solution and carry out aerobic, two sections combined fermentations of anaerobism, fs is the aerobic fermentation stage, control pH5.5～6.0, keeping oxyty is 60～90%, temperature is controlled at 30～32 ℃, aerobic cultivation 10～20 hours, when cell concn is that optical density(OD) (O.D.) is when reaching 15～30, change the subordinate phase anaerobically fermenting over to, anaerobism is cultivated until fermentation ends, the anaerobic stages temperature is controlled at 35～38 ℃, ferments 40～50 hours, promptly obtains γ-An Jidingsuan.
Wherein, described plate culture medium comprises the component of following weight per-cent: corn cob hydrolyzed solution 20～30%, and yeast extract paste 0.3～0.5%, peptone 0.7～0.9%, corn steep liquor 0.2～0.4%, agar 2%, all the other are water.
Described shake-flask seed substratum comprises the component of following weight per-cent: corn cob hydrolyzed solution 20～30%, and yeast extract paste 0.3～0.5%, peptone 0.7～0.9%, corn steep liquor 0.2～0.4%, all the other are water.
Described seed tank culture base comprises the component of following weight per-cent: corn cob hydrolyzed solution 20～30%, and yeast extract paste 0.2～0.4%, peptone 0.4～0.6%, corn steep liquor 0.1～0.3%, all the other are water.
The present invention can be with fermented liquid through exchange resin absorption after fermentation ends, and concentrated, crystallization get the γ-An Jidingsuan crystal.
γ-An Jidingsuan preparation method of the present invention utilizes corn cob hydrolyzed solution fermentative production γ-An Jidingsuan, and it carries out aerobic, anaerobically fermenting in growth, metabolic different steps to the demand difference of oxygen, various nutritive element, various carbon source, nitrogenous source according to this kind microorganism.Sectional temperature-controlled in the fermenting process, as to take segmentation to regulate and control on the demand to oxygen means make γ-An Jidingsuan metabolism under anaerobic state produce γ-An Jidingsuan.
γ-An Jidingsuan preparation method of the present invention has following beneficial effect:
1) biological fermentation process of the present invention is that utilization is that substrate is produced thalline with the extensive corn cob hydrolyzed solution that contains glucose and wood sugar, and the corn cob hydrolyzed solution is turned waste into wealth, and has reduced environmental pollution, and can effectively reduce the production cost of γ-An Jidingsuan.
2) biotechnology production γ-An Jidingsuan is that L-Glutamic decarboxylase turns to γ-An Jidingsuan with the L-glutamic acid rotating in microorganism cells, and production process is simple, safe, energy-conservation, is a kind of up-and-coming production method.
3) fermentation technique of the present invention adopts aerobic, two sections combined fermentations of anaerobism, sectional temperature-controlled in the fermenting process, on demand, take the means of segmentation regulation and control to oxygen, make γ-An Jidingsuan produce bacterial strain metabolism under anaerobic state and produce γ-An Jidingsuan, sectionalized ventilation and thermophilic fermentation have shortened fermentation period, have reduced cooling water amount, reduce cost, improved the productive rate of γ-An Jidingsuan.
4) production process is simple, safe, energy-conservation, has also avoided the use of the aggressive solvent in the traditional method, has protected environment.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
The preparation process of the corn cob hydrolyzed solution that present embodiment adopted is:
1) corncob acid hydrolysis: with corn cob in the sulphuric acid soln of concentration expressed in percentage by volume 5%, be hydrolyzed acid hydrolysis solution, hydrolysis temperature is 200 ℃, hydrolysis time 300 minutes;
2) solid-liquid separation: the acid hydrolysis solution solid-liquid separation, first liquid glucose and the corncob acid hydrolysis slag of wood sugar is rich in acquisition;
3) acidolysis slag enzymolysis: one or more in usefulness cellulase, zytase or the cellobiase, the corncob acid hydrolysis slag is carried out enzymic hydrolysis, get enzymolysis solution; Enzyme dosage is: every gram acidolysis slag 50PFIU cellulase, 30IU cellobiase, 200PFIU zytase; Control pH is 7.0 in the enzymolysis process, and temperature is 60 ℃, and enzymolysis time is 240 hours;
4) solid-liquid separation: the enzymolysis solution solid-liquid separation obtains being rich in second liquid glucose (being the corn cob hydrolyzed solution) of glucose and is rich in the enzymolysis slag of xylogen.The corn cob hydrolyzed solution contains 8% glucose and 2% wood sugar and a small amount of pectinose.
The γ-An Jidingsuan preparation process of present embodiment is as follows:
Earlier γ-An Jidingsuan being produced bacterial strain breast tyrothricin CGMCC NO.1306 (Lactobacillus brevis CGMCCNO.1306) receives and contains corn cob hydrolyzed solution 20%, yeast extract paste 0.4%, peptone 0.8%, corn steep liquor 0.3%, in the plate culture medium of agar 2%, cultivated 24 hours for 30 ℃.
Adopt dull and stereotyped γ-An Jidingsuan inoculation one of cultivating to encircle to then and contain corn cob hydrolyzed solution 20%, yeast extract paste 0.3%, peptone 0.7%, cultivate in the liquid seed culture medium of corn steep liquor 0.2%, pH is controlled at 5.5, and shaking table was cultivated 20 hours, shaking speed 220rpm, 33 ℃ of culture temperature.
After treating that shake-flask seed is cultivated, inoculate and contain corn cob hydrolyzed solution 30%, yeast extract paste 0.3%, peptone 0.5% ventilates in the seeding tank liquid seed culture medium of corn steep liquor 0.2% and cultivates 33 ℃ of culture temperature, pH is controlled at 5.0, mixing speed 100rpm, sterile air ventilating ratio 1: 0.7 (v/v), culture cycle 15 hours.
Well back being inoculated in by 5% (v/v) inoculum size of seeding tank seed culture contained corn cob hydrolyzed solution 20%, yeast extract paste 0.3%, peptone 0.2%, corn steep liquor 0.5%, MgSO
47H
2O0.002%, MnSO
44H
2O 0.005%, KH
2PO
40.03%, Soduxin 0.6%, cultivating in the 5L fermentor tank of L-Sodium Glutamate 6%, is the aerobic fermentation stage in the fs, and sterile air is that 1: 0.8 rate of venting flows in the fermention medium of fermentor tank with ventilating ratio, mixing speed is 300rpm, keeping oxyty (DO) is 65～75%, and temperature is controlled at 31 ℃, aerobic cultivation 18 hours, when cell concn is an optical density(OD) (O.D.) when reaching 28, change the subordinate phase fermentation over to; In subordinate phase is the anaerobically fermenting stage, and anaerobism is cultivated until fermentation ends, and temperature is controlled at 36 ℃.Add ammoniacal liquor or sodium hydroxide fermentation system pH is controlled at 5.8, fermented 40 hours, the γ-An Jidingsuan productive rate reaches 3.7%.
After the fermentation ends, through the absorption of D061 type exchange resin, the optimal adsorption condition is pH=4.0 with fermented liquid, 40 ℃ of temperature, and equilibrium adsorption time 17min, adsorptive capacity is 0.83mol/L.The flow velocity of sample is 1BV/h on the gamma-aminobutyric acid fermentation.The eluent ammonia concn is 1.2mol/L, and flow velocity is 1BV/h.Concentrate, after the alcohol precipitation crystallization, can get the γ-An Jidingsuan crystal through Rotary Evaporators, its rate of recovery reaches 80%.
Embodiment 2
The γ-An Jidingsuan preparation process of present embodiment is as follows:
Earlier γ-An Jidingsuan being produced bacterial strain breast tyrothricin CGMCC NO.1306 (Lactobacillus brevis CGMCCNO.1306) receives and contains corn cob hydrolyzed solution 20%, yeast extract paste 0.4%, peptone 0.8%, corn steep liquor 0.3%, in the plate culture medium of agar 2%, cultivated 26 hours for 30 ℃.
Adopt dull and stereotyped γ-An Jidingsuan inoculation one of cultivating to encircle to then and contain corn cob hydrolyzed solution 25%, yeast extract paste 0.4%, peptone 0.8%, cultivate in the liquid seed culture medium of corn steep liquor 0.2%, pH is controlled at 5.2, and shaking table was cultivated 20 hours, shaking speed 220rpm, 33 ℃ of culture temperature.
After treating that shake-flask seed is cultivated, inoculate and contain corn cob hydrolyzed solution 30%, yeast extract paste 0.3%, peptone 0.5% ventilates in the seeding tank liquid seed culture medium of corn steep liquor 0.2% and cultivates 30 ℃ of culture temperature, pH is controlled at 5.5, mixing speed 75rpm, sterile air ventilating ratio 1: 0.8 (v/v), culture cycle 16 hours.
Be inoculated in after the seeding tank seed culture is good and contain corn cob hydrolyzed solution 20%, yeast extract paste 0.3%, peptone 0.2%, corn steep liquor 0.5%, MgSO
47H
2O 0.001%, MnSO
44H
2O0.005%, KH
2PO
40.04%, Soduxin 0.6%, cultivating in the 5L fermentor tank of L-Sodium Glutamate 6%, is the aerobic fermentation stage in the fs, and sterile air is that 1: 0.9 rate of venting flows in the fermention medium of fermentor tank with ventilating ratio, mixing speed is 300rpm, keeping oxyty (DO) is 60～70%, and temperature is controlled at 31 ℃, aerobic cultivation 18 hours, when cell concn is an optical density(OD) (O.D.) when reaching 28, change the subordinate phase fermentation over to; In subordinate phase is the anaerobically fermenting stage, and anaerobism is cultivated until fermentation ends, and temperature is controlled at 36 ℃.Add ammoniacal liquor or sodium hydroxide fermentation system pH is controlled at 5.7, fermented 50 hours, the γ-An Jidingsuan productive rate reaches 3.9%.
After the fermentation ends, through the absorption of D061 type exchange resin, the optimal adsorption condition is pH=4.0 with fermented liquid, 40 ℃ of temperature, and equilibrium adsorption time 17min, adsorptive capacity is 0.83mol/L.The flow velocity of sample is 1BV/h on the gamma-aminobutyric acid fermentation.The eluent ammonia concn is 1.2mol/L, and flow velocity is 1BV/h.Concentrate, after the alcohol precipitation crystallization, can get the γ-An Jidingsuan crystal through Rotary Evaporators, its rate of recovery reaches 80%.
Embodiment 3
The γ-An Jidingsuan preparation process of present embodiment is as follows:
Earlier γ-An Jidingsuan being produced bacterial strain breast tyrothricin CGMCC NO.1306 (Lactobacillus brevis CGMCCNO.1306) receives and contains corn cob hydrolyzed solution 25%, yeast extract paste 0.3%, peptone 0.8%, corn steep liquor 0.3%, in the plate culture medium of agar 2%, cultivated 18 hours for 30 ℃.
Adopt dull and stereotyped γ-An Jidingsuan inoculation one of cultivating to encircle to then and contain corn cob hydrolyzed solution 20%, yeast extract paste 0.3%, peptone 0.7%, cultivate in the liquid seed culture medium of corn steep liquor 0.4%, pH is controlled at 5.5, and shaking table was cultivated 24 hours, shaking speed 180rpm, 30 ℃ of culture temperature.
After treating that shake-flask seed is cultivated, inoculate and contain corn cob hydrolyzed solution 25%, yeast extract paste 0.2%, peptone 0.5% ventilates in the seeding tank liquid seed culture medium of corn steep liquor 0.2% and cultivates 33 ℃ of culture temperature, pH is controlled at 5.2, mixing speed 80rpm, sterile air ventilating ratio 1: 0.8 (v/v), culture cycle 15 hours.
Be inoculated in after the seeding tank seed culture is good and contain corn cob hydrolyzed solution 25%, yeast extract paste 0.2%, peptone 0.1%, corn steep liquor 0.5%, MgSO
47H
2O 0.001%, MnSO
44H
2O0.006%, KH
2PO
40.05%, Soduxin 0.6%, cultivating in the 5L fermentor tank of L-Sodium Glutamate 6%, is the aerobic fermentation stage in the fs, and sterile air is that 1: 0.8 rate of venting flows in the fermention medium of fermentor tank with ventilating ratio, mixing speed is 500rpm, keeping oxyty (DO) is 70～80%, and temperature is controlled at 31 ℃, aerobic cultivation 10 hours, when cell concn is an optical density(OD) (O.D.) when reaching 20, change the subordinate phase fermentation over to; In subordinate phase is the anaerobically fermenting stage, and anaerobism is cultivated until fermentation ends, and temperature is controlled at 37 ℃.Add ammoniacal liquor or sodium hydroxide fermentation system pH is controlled at 5.8, fermented 40 hours, the γ-An Jidingsuan productive rate reaches 3.5%.
After the fermentation ends, fermented liquid is adsorbed through D061 type exchange resin, the optimal adsorption condition is pH=4.0,40 ℃ of temperature, equilibrium adsorption time 17min, adsorptive capacity is that the flow velocity of sample on the 0.83mol/L. gamma-aminobutyric acid fermentation is that 1BV/h. eluent ammonia concn is 1.2mol/L, flow velocity be 1BV/h. through Rotary Evaporators concentrate, after the alcohol precipitation crystallization, can get the γ-An Jidingsuan crystal, its rate of recovery reaches 80%.
Embodiment 4
The γ-An Jidingsuan preparation process of present embodiment is as follows:
Earlier γ-An Jidingsuan being produced bacterial strain breast tyrothricin CGMCC NO.1306 (Lactobacillus brevis CGMCCNO.1306) receives and contains corn cob hydrolyzed solution 30%, yeast extract paste 0.4%, peptone 0.9%, corn steep liquor 0.2%, in the plate culture medium of agar 2%, cultivated 20 hours for 30 ℃.
Adopt dull and stereotyped γ-An Jidingsuan inoculation one of cultivating to encircle to then and contain corn cob hydrolyzed solution 25%, yeast extract paste 0.5%, peptone 0.9%, cultivate in the liquid seed culture medium of corn steep liquor 0.3%, pH is controlled at 5.0, and shaking table was cultivated 24 hours, shaking speed 200rpm, 33 ℃ of culture temperature.
After treating that shake-flask seed is cultivated, inoculate and contain corn cob hydrolysis sugar 20%, yeast extract paste 0.3%, peptone 0.5% ventilates in the seeding tank liquid seed culture medium of corn steep liquor 0.2% and cultivates 33 ℃ of culture temperature, pH is controlled at 5.4, mixing speed 100rpm, sterile air ventilating ratio 1: 0.6 (v/v), culture cycle 20 hours.
Be inoculated in after the seeding tank seed culture is good and contain corn cob hydrolyzed solution 25%, yeast extract paste 0.4%, peptone 0.3%, corn steep liquor 0.4%, MgSO
47H
2O 0.002%, MnSO
44H
2O0.005%, KH
2PO
40.002%, Soduxin 0.6%, cultivating in the 5L fermentor tank of L-Sodium Glutamate 6%, is the aerobic fermentation stage in the fs, and sterile air is that 1: 1.2 rate of venting flows in the fermention medium of fermentor tank with ventilating ratio, mixing speed is 300rpm, keeping oxyty (DO) is 75～85%, and temperature is controlled at 31 ℃, aerobic cultivation 15 hours, when cell concn is an optical density(OD) (O.D.) when reaching 15, change the subordinate phase fermentation over to; In subordinate phase is the anaerobically fermenting stage, and anaerobism is cultivated until fermentation ends, and temperature is controlled at 36 ℃.Add ammoniacal liquor or sodium hydroxide fermentation system pH is controlled at 5.6, fermented 45 hours, the γ-An Jidingsuan productive rate reaches 3.97%.
After the fermentation ends, through the absorption of D061 type exchange resin, the optimal adsorption condition is pH=4.0 with fermented liquid, 40 ℃ of temperature, and equilibrium adsorption time 17min, adsorptive capacity is 0.83mol/L.The flow velocity of sample is 1BV/h on the gamma-aminobutyric acid fermentation.The eluent ammonia concn is 1.2mol/L, and flow velocity is 1BV/h.Concentrate, after the alcohol precipitation crystallization, can get the γ-An Jidingsuan crystal through Rotary Evaporators, its rate of recovery reaches 80%.
Embodiment 5
The γ-An Jidingsuan preparation process of present embodiment is as follows:
Earlier γ-An Jidingsuan being produced bacterial strain breast tyrothricin CGMCC NO.1306 (Lactobacillus brevis CGMCCNO.1306) receives and contains corn cob hydrolyzed solution 20%, yeast extract paste 0.4%, peptone 0.7%, corn steep liquor 0.4%, in the plate culture medium of agar 2%, cultivated 22 hours for 30 ℃.
Adopt dull and stereotyped γ-An Jidingsuan inoculation one of cultivating to encircle to then and contain corn cob hydrolyzed solution 30%, yeast extract paste 0.5%, peptone 0.9%, cultivate in the liquid seed culture medium of corn steep liquor 0.3%, pH is controlled at 5.4, and shaking table was cultivated 22 hours, shaking speed 200rpm, 32 ℃ of culture temperature.
After treating that shake-flask seed is cultivated, inoculate and contain corn cob hydrolyzed solution 30%, yeast extract paste 0.4%, peptone 0.6% ventilates in the seeding tank liquid seed culture medium of corn steep liquor 0.2% and cultivates 33 ℃ of culture temperature, pH is controlled at 5.2, mixing speed 90rpm, sterile air ventilating ratio 1: 0.6 (v/v), culture cycle 15 hours.
Be inoculated in after the seeding tank seed culture is good and contain corn cob hydrolyzed solution 26%, yeast extract paste 0.3%, peptone 0.2%, corn steep liquor 0.6%, MgSO
47H
2O 0.002%, MnSO
44H
2O0.005%, KH
2PO
40.07%, Soduxin 0.5%, cultivating in the 5L fermentor tank of L-Sodium Glutamate 7%, is the aerobic fermentation stage in the fs, and sterile air is that 1: 0.8 rate of venting flows in the fermention medium of fermentor tank with ventilating ratio, mixing speed is 400rpm, keeping oxyty (DO) is 65～75%, and temperature is controlled at 31 ℃, aerobic cultivation 20 hours, when cell concn is an optical density(OD) (O.D.) when reaching 30, change the subordinate phase fermentation over to; In subordinate phase is the anaerobically fermenting stage, and anaerobism is cultivated until fermentation ends, and temperature is controlled at 36 ℃. add ammoniacal liquor or sodium hydroxide fermentation system pH is controlled at 5.7, fermented 50 hours, the γ-An Jidingsuan productive rate reaches 3.4%.
After the fermentation ends, through the absorption of D061 type exchange resin, the optimal adsorption condition is pH=4.0 with fermented liquid, 40 ℃ of temperature, and equilibrium adsorption time 17min, adsorptive capacity is 0.83mol/L.The flow velocity of sample is 1BV/h on the gamma-aminobutyric acid fermentation.The eluent ammonia concn is 1.2mol/L, and flow velocity is 1BV/h.Concentrate, after the alcohol precipitation crystallization, can get the γ-An Jidingsuan crystal through Rotary Evaporators, its rate of recovery reaches 80%.
Embodiment 6
The γ-An Jidingsuan preparation process of present embodiment is as follows:
Earlier γ-An Jidingsuan being produced bacterial strain breast tyrothricin CGMCC NO.1306 (Lactobacillus brevis CGMCC NO.1306) receives and contains corn cob hydrolyzed solution 30%, yeast extract paste 0.5%, peptone 0.8%, corn steep liquor 0.3%, in the plate culture medium of agar 2%, cultivated 26 hours for 30 ℃.
Adopt dull and stereotyped γ-An Jidingsuan inoculation one of cultivating to encircle to then and contain corn cob hydrolyzed solution 20%, yeast extract paste 0.3%, peptone 0.7%, cultivate in the liquid seed culture medium of corn steep liquor 0.4%, pH is controlled at 5.5, and shaking table was cultivated 20 hours, shaking speed 220rpm, 30 ℃ of culture temperature.
After treating that shake-flask seed is cultivated, inoculate and contain corn cob hydrolyzed solution 30%, yeast extract paste 0.2%, peptone 0.5% ventilates in the seeding tank liquid seed culture medium of corn steep liquor 0.1% and cultivates 32 ℃ of culture temperature, pH is controlled at 5.5, mixing speed 100rpm, sterile air ventilating ratio 1: 0.7 (v/v), culture cycle 18 hours.
Be inoculated in after the seeding tank seed culture is good and contain corn cob hydrolyzed solution 27%, yeast extract paste 0.3%, peptone 0.2%, corn steep liquor 0.5%, MgSO
47H
2O 0.002%, MnSO
44H
2O0.005%, KH
2PO
40.05%, Soduxin 0.7%, cultivating in the 5L fermentor tank of L-Sodium Glutamate 4%, is the aerobic fermentation stage in the fs, and sterile air is that 1: 0.9 rate of venting flows in the fermention medium of fermentor tank with ventilating ratio, mixing speed is 350rpm, keeping oxyty (DO) is 80～90%, and temperature is controlled at 31 ℃, aerobic cultivation 18 hours, when cell concn is an optical density(OD) (O.D.) when reaching 25, change the subordinate phase fermentation over to; In subordinate phase is the anaerobically fermenting stage, and anaerobism is cultivated until fermentation ends, and temperature is controlled at 36 ℃.Add ammoniacal liquor or sodium hydroxide fermentation system pH is controlled at 5.8, fermented 40 hours, the γ-An Jidingsuan productive rate reaches 3.84%.
After the fermentation ends, through the absorption of D061 type exchange resin, the optimal adsorption condition is pH=4.0 with fermented liquid, 40 ℃ of temperature, and equilibrium adsorption time 17min, adsorptive capacity is 0.83mol/L.The flow velocity of sample is 1BV/h on the gamma-aminobutyric acid fermentation.The eluent ammonia concn is 1.2mol/L, and flow velocity is 1BV/h.Concentrate, after the alcohol precipitation crystallization, can get the γ-An Jidingsuan crystal through Rotary Evaporators, its rate of recovery reaches 80%.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the preparation method of a γ-An Jidingsuan is characterized in that, it comprises the steps:
Will be in fermention medium through the γ-An Jidingsuan production inoculation of cultivating, carry out aerobic, two sections combined fermentations of anaerobism, fs is the aerobic fermentation stage, control pH5.5～6.0, keeping oxyty is 60～90%, temperature is controlled at 30～32 ℃, aerobic cultivation 10～20 hours, when cell concn reaches 15～30, change the subordinate phase anaerobically fermenting over to, anaerobism is cultivated until fermentation ends, and the anaerobic stages temperature is controlled at 35～38 ℃, fermented 40～50 hours, and obtained γ-An Jidingsuan.
2. the preparation method of γ-An Jidingsuan as claimed in claim 1 is characterized in that, it is newborn tyrothricin CGMCC NO.1306 (Lactobacillus brevis CGMCC NO.1306) that described γ-An Jidingsuan is produced bacterial strain.
3. the preparation method of γ-An Jidingsuan as claimed in claim 1 or 2 is characterized in that, in the fs, by being 1 with sterile air with ventilating ratio: the rate of venting of (0.8～1.2) flows in the fermention medium, and mixing speed is 300～500rpm.
4. as the preparation method of any described γ-An Jidingsuan of claim 1-3, it is characterized in that the carbon source of described fermention medium is one or more and the L-Sodium Glutamate in corn cob hydrolyzed solution, straw hydrolyzed solution or the bagasse hydrolysis sugar; Nitrogenous source is yeast extract paste, peptone, corn steep liquor, urea, ammonium sulfate, one or more in wheat bran hydrolyzed solution or the soybean meal hydrolysate.
5. as the preparation method of any described γ-An Jidingsuan of claim 1-4, it is characterized in that the pH value of described fermention medium is regulated one or more in employing ammoniacal liquor, sodium hydroxide, yellow soda ash, lime carbonate or the calcium hydroxide.
6. as the preparation method of any described γ-An Jidingsuan of claim 1-5, it is characterized in that, described fermentation tank culture medium comprises the component of following weight per-cent: corn cob hydrolyzed solution 20～30%, yeast extract paste 0.2～0.4%, peptone 0.1～0.3%, corn steep liquor 0.4～0.6%, MgSO
47H
2O 0.001～0.003%, MnSO
44H
2O 0.004～0.006%, KH
2PO
40.02～0.07%, Soduxin 0.5～0.7%, L-Sodium Glutamate 5～7%, all the other are water.
7. the preparation method of γ-An Jidingsuan as claimed in claim 1 is characterized in that, before carrying out fermentation culture, described γ-An Jidingsuan is produced bacterial strain and carried out following cultivation earlier: comprise the steps:
1) the dull and stereotyped cultivation: adopting the corn cob hydrolyzed solution to add nitrogenous source is plate culture medium, γ-An Jidingsuan is produced bacterial strain receive in the plate culture medium that contains the corn cob hydrolyzed solution, cultivates 18～26 hours for 30 ℃;
2) shake-flask seed is cultivated: the γ-An Jidingsuan production bacterial strain that flat board is cultivated is received in the shake-flask seed substratum that contains the corn cob hydrolyzed solution, cultivated 20～24 hours for 30～33 ℃, pH is controlled at 5.0～5.5, shaking speed 180～220rpm;
3) seeding tank seed culture: the γ-An Jidingsuan of shake-flask seed culture medium culturing is produced bacterial strain receive in the seed tank culture base that contains the corn cob hydrolyzed solution, inoculum size is 5～10%, cultivated 15～20 hours for 30～33 ℃, pH is controlled at 5.0～5.5, mixing speed 75～100rpm, ventilating ratio 1: (0.6～0.8).
8. the preparation method of γ-An Jidingsuan as claimed in claim 7, it is characterized in that, described plate culture medium comprises the component of following weight per-cent: corn cob hydrolyzed solution 20～30%, yeast extract paste 0.3～0.5%, peptone 0.7～0.9%, corn steep liquor 0.2～0.4%, agar 2%, all the other are water.
9. the preparation method of γ-An Jidingsuan as claimed in claim 7 is characterized in that, described shake-flask seed substratum comprises the component of following weight per-cent: corn cob hydrolyzed solution 20～30%, yeast extract paste 0.3～0.5%, peptone 0.7～0.9%, corn steep liquor 0.2～0.4%, all the other are water.
10. the preparation method of γ-An Jidingsuan as claimed in claim 7 is characterized in that, described seed tank culture base comprises the component of following weight per-cent: corn cob hydrolyzed solution 20～30%, yeast extract paste 0.2～0.4%, peptone 0.4～0.6%, corn steep liquor 0.1～0.3%, all the other are water.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101926412A (en) * | 2010-09-30 | 2010-12-29 | 湖北新生源生物工程股份有限公司 | Production method of compound amino acid feed additive rich in gamma-aminobutyric acid (GABA) |
| CN103031322A (en) * | 2011-09-30 | 2013-04-10 | 浙江大学宁波理工学院 | Glutamate decarboxylase (GAD) thermally-stable variant G311P gene and application thereof |
| CN103773731A (en) * | 2014-01-03 | 2014-05-07 | 浙江大学宁波理工学院 | Method for improving apparent catalytic activity of glutamic acid decarboxylase recombinant engineering bacteria |
| CN107439731A (en) * | 2016-06-01 | 2017-12-08 | 周炳志 | The manufacture method of tealeaves containing gamma-aminobutyric acid |
| CN108208590A (en) * | 2017-12-29 | 2018-06-29 | 涂慧燕 | A kind of germinated rice rice flour of high gamma-aminobutyric acid and phytic acid and preparation method thereof |
| CN109699917A (en) * | 2019-02-22 | 2019-05-03 | 江汉大学 | A kind of method that the preparation of multi-cultur es combined ferment is rich in γ-aminobutyric acid soy food product |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215582B (en) * | 2007-12-28 | 2010-12-15 | 江南大学 | Method for producing succinic acid by fermenting straw raw material |
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2009
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101926412A (en) * | 2010-09-30 | 2010-12-29 | 湖北新生源生物工程股份有限公司 | Production method of compound amino acid feed additive rich in gamma-aminobutyric acid (GABA) |
| CN101926412B (en) * | 2010-09-30 | 2012-11-07 | 湖北新生源生物工程股份有限公司 | Production method of compound amino acid feed additive rich in gamma-aminobutyric acid (GABA) |
| CN103031322A (en) * | 2011-09-30 | 2013-04-10 | 浙江大学宁波理工学院 | Glutamate decarboxylase (GAD) thermally-stable variant G311P gene and application thereof |
| CN103031322B (en) * | 2011-09-30 | 2014-10-22 | 浙江大学宁波理工学院 | Glutamate decarboxylase (GAD) thermally-stable variant G311P gene and application thereof |
| CN103773731A (en) * | 2014-01-03 | 2014-05-07 | 浙江大学宁波理工学院 | Method for improving apparent catalytic activity of glutamic acid decarboxylase recombinant engineering bacteria |
| CN107439731A (en) * | 2016-06-01 | 2017-12-08 | 周炳志 | The manufacture method of tealeaves containing gamma-aminobutyric acid |
| CN108208590A (en) * | 2017-12-29 | 2018-06-29 | 涂慧燕 | A kind of germinated rice rice flour of high gamma-aminobutyric acid and phytic acid and preparation method thereof |
| CN109699917A (en) * | 2019-02-22 | 2019-05-03 | 江汉大学 | A kind of method that the preparation of multi-cultur es combined ferment is rich in γ-aminobutyric acid soy food product |
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