CN101654689B - Method for biologically synthesizing gamma-aminobutyric acid by pediococcus pentosaceus - Google Patents
Method for biologically synthesizing gamma-aminobutyric acid by pediococcus pentosaceus Download PDFInfo
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Abstract
The invention discloses a method for biologically synthesizing gamma-aminobutyric acid, which uses the pediococcus pentosaceus as the strain, and converts the externally added glutamic acid or monosodium glutamate through fermentation engineering technology, cell transformation method and enzyme engineering technology to produce high content gamma-aminobutyric acid (conversion liquid gamma-aminobutyric acid content reaches 1-30%). The method belongs to the biological technical field, doesn't need high temperature, high pressure reaction condition or toxic harmful raw materials, has the advantages of high maneuverability and little by-product, and the pediococcus pentosaceus is a food grade microorganism with high safety, thus applying to industries such as chemical industry, food, forage or medicine after purifying.
Description
Technical field
The present invention relates to utilize the method for the synthetic γ-An Jidingsuan of microorganism, particularly utilize the method for the synthetic γ-An Jidingsuan of Pediococcus pentosaceus (Pediococcus pentosaceus), belong to biological technical field.
Background technology
γ-An Jidingsuan (γ-Aminobutyric acid, GABA), be aminobutyric acid again, it is the natural amino acid that a kind of nonprotein is formed, it is a kind of main inhibitory nerve mediator of mammalian central nervous system, have the important physical function, as bring high blood pressure down, the brain function is calmed the nerves, is improved in diuresis, analgesia, promote the brain vigor, promote long-term memory, trophic nerve cell, improve climacteric syndrome etc.GABA will cause diseases such as epilepsy, Parkinson when the brain long-term lacking.Simultaneously GABA is also relevant with brain aging, and its shortage will cause the elderly " ear is not clever, order not clear ".In addition, GABA can promote the ovum ability of wearing of sperm, improves rate of fertilization, be used to cover up or reduce the material with unpleasant taste the unpleasant flavor impression purposes and can improve efficiency of feed utilization and day weight gain.γ-An Jidingsuan is just extensively applied to industries such as medicine, health care of food, chemical industry and agricultural.The production of GABA mainly can be by chemosynthesis and two approach of biosynthesizing.The chemosynthesis reaction condition is violent, and the chemical solvents of employing has toxicity and corrodibility, and by product is many, lacks security, is mainly used in chemical industry; Biological synthesis process has advantages such as mild condition, environmental pollution are relatively low, security height than chemical synthesis.In addition,, therefore utilize microorganisms producing GABA, not limited by resource, environment and spatial, have significant advantage because microorganism has that fast growth, growth conditions are simple, metabolic process is special and the characteristics such as wide that distribute.
At present there have been some to utilize the report of the microorganisms producing GABA of different genera.
Lu Zhaoxin, Yang Shengyuan etc. disclose a kind of production method of γ-An Jidingsuan in Chinese patent (patent No. ZL 200510040758.9), it is to be bacterial classification with saliva chain coccus thermophilous subspecies (Streptococcus thermophilus), act on L-glutamic acid, glutaminate, contain the material of L-glutamic acid or glutaminate, make the α-carboxyl generation decarboxylation of L-glutamic acid, thereby generate γ-An Jidingsuan.
Wu Tianxiang etc. disclose the method that solid state fermentation prepares γ-An Jidingsuan in Chinese patent (publication number CN101240301), comprise the steps: at first to be that raw material screening goes out monascus specie with the fermented bean curd; Then monascus MP1104 bacterial classification is placed and cultivate 7d on the slant medium, make actication of culture; Fermented bacterium is transferred in the substratum then, under 30 ℃ of temperature, rotating speed 150r/min the activated spawn shaking table is cultivated 2d, preparation ferment-seeded, preferred solid state fermentation conditions and substratum; At last bacterial classification is cultivated under preferred substratum and the culture condition in previous step is rapid and produced GABA.This method is a fermentation raw material with the rice, is bacterial classification with the monascus, has edible safety, can be used as protective foods and directly eats; The GABA output and the purity of this method are all high, and under optimal conditions of fermentation and substratum, GABA output can reach 0.35mg/g by initial 0.21mg/g, and the purity of the finished product can reach 45%.
Burnt celebrating just waits the enzymatic conversion preparation method that discloses a kind of γ-An Jidingsuan in Chinese patent (patent No. ZL 200410064813.3), this preparation method uses L-L-glutamic acid and two kinds of mixing acid acidic amino acids of L-aspartic acid as raw material, the somatic cells that will have the Escherichia Escherichia coli AS1.505 of high vigor L-L-Glutamic decarboxylase mixes with the conversion fluid that contains L-L-glutamic acid and L-aspartic acid mixture, carry out enzymatic reaction under 28-45 ℃, separate converted product with isoelectric point crystallizing method or isoelectric point crystallizing with the method that ion exchange resin combines then, obtain highly purified γ-An Jidingsuan and L-aspartic acid.This method has solved the difficult problem of two kinds of acidic mixed amino acid high efficiency separation, has obtained the higher γ-An Jidingsuan of added value, and advantage such as it is cheap, easy and simple to handle to have a cost of material, and transformation time is short, and production cost is low.
The happy grade of plum discloses a kind of method of biosynthesizing γ-An Jidingsuan in Chinese patent (patent No. ZL 200510049187.5), it is characterized in that: deposit number is the short lactobacillus (Lactobacillus brevis) of CGMCC NO.1306, after the agar slant culture-medium activation, transfer in GYP seed culture medium or MRS seed culture medium, cultivate after 10～30 hours, inoculum size with 0.5%～5% is inoculated in GYP or the MRS fermention medium, under 25 ℃～35 ℃, leave standstill and cultivate 48h～120h, promptly get the fermented liquid of mycetome, the thalline centrifugation is collected; Thalline after centrifugal is again with the deionized water wash of the bacterium of going out, get 0.25～2g wet thallus, be suspended in citric acid-Sodium phosphate dibasic buffer system of 15～50mL, L-Sodium Glutamate content is 5mM～60mM, reacted the centrifugal solution that contains γ-An Jidingsuan that promptly gets of reaction solution 1～10 hour.The happy grade of plum also discloses a kind of method of the pH of control fermentative production γ-An Jidingsuan in Chinese patent (patent No. ZL 200510049187.5).It is characterized in that: deposit number is the short lactobacillus (Lactobacillus brevis) of CGMCC NO.1306, after the agar slant culture-medium activation, transfer in the GYP seed culture medium, cultivate after 25～35 hours, inoculum size with 5～10% is inoculated in the fermentor tank, the fermentor tank liquid amount is 1～3L, and mixing speed is 50～150r/min, leaves standstill cultivation under 30 ℃, it is carried out pH control fermentation, about 25～40 hours of fermentation culture treats that thalli growth enters stationary phase, after the pH value is gone up, Continuous Flow adds the hydrochloric acid of 1～3mol/L, fermention medium pH is controlled at 5.0～5.6, continues to cultivate about 40～60h, promptly gets the fermented liquid that contains γ-An Jidingsuan.
Guo Xiaofeng etc. disclose a kind of production method that relates to the food that contains γ-An Jidingsuan in Chinese patent (patent publication No. CN101102683), it comprises makes yeast or its handled thing act on sugar and/or carbohydrate metabolism intermediate, or act on sugar or carbohydrate metabolism intermediate and L-glutamic acid or its salt, wherein, above-mentioned yeast has the ability of producing γ-An Jidingsuan in the presence of sugar or carbohydrate metabolism intermediate by fermentation reaction.
Jiang Donghua etc. disclose a kind of high yield gamma-reanal monascus ruber Mr-5-5 bacterial strain and screening method and purposes in Chinese patent (patent publication No. CN101302480).The high yield GABA red monascus of this invention (Monascus ruber Mr-5) bacterial strain, its deposit number is: CCTCC NO:M208043 is to preservation: Chinese typical culture collection center.Also disclose screening method and the purposes and the synthetic method that is used for γ-An Jidingsuan of above-mentioned red monascus Mr-5 bacterial strain in addition, contained the γ-An Jidingsuan of 6～9g/L in the fermented liquid of the method gained of the synthetic γ-An Jidingsuan of employing biological process.
Cui Xiaojun etc. disclose a kind of method of biosynthetic gamma-aminobutyric acid preparation in Chinese patent (patent publication No. CN101311273), this product contains by weight percentage: 5% to 60% γ-An Jidingsuan.The preparation method of above-mentioned biosynthetic gamma-aminobutyric acid preparation carries out according to the following steps: at first the streptococcus acidi lactici kind is inoculated into 250mL by glucose, the corn starch, the defatted soybean meal powder, the ferment-seeded substratum that monosodium glutamate is formed, form fermented liquid, fermented liquid is introduced centrifugal formation clear liquid in the supercentrifuge, with clear liquid under 40 ℃, add the 250mg/L chitosan, stir flocculation, to pass through flame filter press through the fermented liquid to be filtered of flocculation, obtain cleaner liquid, cleaner liquid carries out ion-exchange through cation resin exchange bed, treat that ion exchange resin is saturated after, the deionized water wash-out, with the whole wash-outs of L-glutamic acid, extract γ-An Jidingsuan with the ammoniacal liquor wash-out then.
Cao Yusheng etc. disclose a kind of short lactobacillus of highly producing gamma-aminobutyric acid in Chinese patent (patent publication No. CN101333508), its feature and processing method step are: through being accredited as Lactobacillus brevis (short lactobacillus), and national culture presevation number: Lactobacillus brevisCCTCCM 208054.To be preserved in the short lactobacillus of MRS agar slant, transfer in the MRS liquid nutrient medium, activated after, be inoculated in the MRSG liquid nutrient medium with the inoculum size of 2-5%, cultivate 60-90h in 25-30 ℃, the γ-An Jidingsuan in the fermented liquid reaches 50-145mM.Cao Yusheng etc. also disclose a kind of method of utilizing short lactobacillus to prepare γ-An Jidingsuan in Chinese patent (patent publication No. CN101333548), its processing method step is: 1. after utilizing the MRS liquid nutrient medium with the short lactobacillus activation, inoculum size with 5% is inoculated in the MRSG fermention medium, cultivate 40-60h, 4 ℃ of centrifugal collection thalline for 34 ℃; 2. utilize 2 rear overhangs of stroke-physiological saline solution washing in the acetate buffer solution that contains 10-100mM Sodium Glutamate, pH5.2,34 ℃ of reaction 1～8h are the solution that contains γ-An Jidingsuan after centrifugal.
Zhao Jinglian etc. are at digest (biotechnology journal, 1989,5 (2): reported 124-128) with calcium alginate embedded method Bacillus coli cells is made immobilized cell, carried out rhythmic reaction, the reaction of continuously stirring formula and the reaction of continuous pillar with 1% glutamic acid solution and produce GABA.Rhythmic reaction 5h transformation efficiency has reached 100%; The continuously stirring formula is reflected in the triangular flask reactor carries out, and with flow velocity input substrate solution and the output-response liquid of 6ml/h, transformation efficiency reaches 85%; Carry out in the column reactor continuously, control flow velocity 12ml/h, transformation efficiency reaches 95%.
Your equality of chapter is at digest (Changsha Institute of Electric Power Engineering journal (natural science edition), 1998,13 (4): reported 433-435) with calcium alginate embedded method Bacillus coli cells is made immobilized cell, waste liquid behind road, the back sodium glutamate mother liquid extraction L-glutamic acid is transformed production GABA, obtained GABA content and reached 98.94%, yield is 49.65%.
(Biosci.Biotechnol.Biochem., 2000,64 (3): introduced the variation to GABA in the Koji making 617-619), GABA content has reached 120 μ g/g to Kono I etc. at digest.
Wang JJ etc. digest (J Ind Microbiol Biotechnol, 2003, reported and utilized Monascus purpureus NTU601 to carry out solid fermentation that GABA content has reached 5004mg/kg in 30:669-676).
(J Ind Microbiol Biotechnol, 2003,30 (1): reported 41-46) that employing Monascus purpureusCCRC31615 carries out solid fermentation, GABA content has reached 1200mg/kg to Su YC etc. at digest.
Nomura M etc. digest (J Dairy Sci., 1998, introduced in 81:1486-1491) and from produce caseic bacterial strain, be separated to a strain Lactococcus lactis 01-7, be used for cheese production, the content of caseic GABA has reached 383mg/kg.
Permitted to build up the Army and in its doctorate paper (Southern Yangtze University, in February, 2004), reported the Lactococcus lactis bacterial strain that from milk-acid bacteria, has screened high yield GABA, 25L jar fermentation 72h, the GABA of fermented liquid has reached 250mg/100ml.
Liu Qing etc. are in digest (amino acid and Biological resources, 2004,26 (1): also screening of high yield GABA milk-acid bacteria and fermentation condition are reported that the GABA in the fermented liquid reaches 3.1g/l 40-43).
Yokoyama S etc. are at digest (Journal of Bioscience and Bioengineering, 2002,93 (1): reported 95-97) and utilized Lactobacillus brevis IFO-12005 that vinasse are fermented, the content of GABA has reached 10.18mM, obtain GABA solution preferably by centrifugal, flocculation, decolouring and deodorization processing, can be used for Food fortification GABA.
The generation of liking to delay high digest (food と science, 2001, No.8 has reported in 81-85) and has adopted Lactobacillus plantarum to utilize the substratum fermentative production GABA that contains the rice bran extract, has reached 5% at dry powder content.
Komatsuzaki N etc. digest (Food Microbiology, 2005, reported that being separated to Lactobacillus paracasei from the japanese traditional leavened food is used for GABA production in 22:497-504), GABA concentration has reached 302mM.
Takahashi T etc. are at digest (Journal of Bioscience and Bioengineering, 2004,97 (6): report has screened GABA transaminase and succinic semialdehyde dehydrogenase defective mutant bacterial strain GAB7-1 and the GAB7-2 of Saccharomyces cerevisiae UT-1 412-418), GABA concentration has reached 0.4mM and 0.42mM respectively in its fermented liquid, has improved 2.0 and 2.1 times respectively than wild strain.
Ijsseldijk etc. are at United States Patent (USP) (United States Patent, US5472718A) disclose in and utilized the sour milk that contains lactobacillus bulgaricus and thermophilus streptococcus to join in the milk, produce cheese, the cheese that is obtained has bigger cellular structure, quality is improved greatly, and has detected micro-GABA therein.
Pediococcus pentosaceus (Pediococcus pentosaceus) is a kind of Gram-positive, amphimicrobian, do not move, can carry out fermentating metabolism produce lactic acid, not aerogenesis, can not reduce nitrate, nonspore-bearing coccus of liquefy gelatin not, be widely used in the fermentation of products such as sausage, livestock product, fishery products, cucumber, green soya bean, soymilk, cheese and silage, had security preferably.Though Pediococcus pentosaceus has obtained to be extensive use of on food and feed, has not yet to see relevant for the report that utilizes Pediococcus pentosaceus biosynthesizing γ-An Jidingsuan.
Summary of the invention
The object of the present invention is to provide the method for utilizing Pediococcus pentosaceus cell or its L-Glutamic decarboxylase to transform the method for external source L-glutamic acid or Sodium Glutamate production γ-An Jidingsuan and utilizing the synthetic γ-An Jidingsuan of Pediococcus pentosaceus submerged fermentation by interpolation external source L-glutamic acid or Sodium Glutamate in fermention medium, can produce high-load γ-An Jidingsuan, reduce production costs greatly, belong to biological technical field.The conversion fluid composition is simple, non-toxic by-products, only need to its GABA simply extract with purifying can be as edible additive.
The inventor is by separating from pickles voluntarily or testing from microbial strains preservation center through buying Pediococcus pentosaceus (Pediococcuspentosaceus), and the result finds that all Pediococcus pentosaceus (Pediococcus pentosaceus) all has higher L-Glutamic decarboxylase activity.Utilize Pediococcus pentosaceus (Pediococcus pentosaceus) cell transformation method and be the production technique of bacterial classification submerged fermentation biosynthesizing γ-An Jidingsuan solution by further having determined with Pediococcus pentosaceus (Pediococcuspentosaceus).
Method of the present invention is:
One, utilizes the direct conversion of substrate of Pediococcus pentosaceus cell that contains the high glutamic acid decarboxylase, suitable buffer condition only need be provided or react pH by dripping soda acid control, composition is few in the transformation system, by product is few, unharmful substance exists, as the starting material safety of food, medicine, reliable.The matter and energy that whole process flow consumes is few, and the waste of generation is few, helps environmental protection.
Two, by Pediococcus pentosaceus is cultivated, centrifugal collection thalline, thalline is carried out fragmentation, suspension liquid or the supernatant liquor behind the recentrifuge got after the fragmentation mix with L-glutamic acid or Sodium Glutamate, react pH and carry out biochemical reaction at a certain temperature at suitable buffer condition or by dripping soda acid control then, thereby obtain γ-An Jidingsuan.Composition is few in the enzymic catalytic reaction system, and by product is few, and unharmful substance exists, as the starting material safety of food, medicine, reliable.The matter and energy that whole process flow consumes is few, and the waste of generation is few, helps environmental protection.
Three, passing through with Pediococcus pentosaceus (Pediococcus pentosaceus) is bacterial classification, by activating step by step and amplifying, the access fermentor tank carries out substep and controls fermentation condition, make high glutamic acid decarboxylase activity cell obtain propagation to greatest extent, act on external source L-glutamic acid or Sodium Glutamate then under suitable fermentation condition, high-content gamma-aminobutyric acid is produced in submerged fermentation.Adopt substep to control fermentation condition and can solve cell proliferation and the inconsistent contradiction of tunning cumulative fermentation condition, the method of γ-An Jidingsuan is produced in submerged fermentation, can produce high-load γ-An Jidingsuan, reduce production costs greatly, simplify zymotechnique.
The molecular weight of the γ-An Jidingsuan of acquisition of the present invention is 103.1, and structural formula is:
The present invention is achieved in that by Pediococcus pentosaceus (Pediococcus pentosaceus) or its L-Glutamic decarboxylase and acts on L-glutamic acid or Sodium Glutamate, makes L-glutamic acid or Sodium Glutamate generation decarboxylation generate γ-An Jidingsuan.Perhaps by being that bacterial classification carries out submerged fermentation synthesis of high content γ-An Jidingsuan with Pediococcus pentosaceus (Pediococcuspentosaceus).
The production method of γ-An Jidingsuan has following form:
1, bacterial classification: Pediococcus pentosaceus (Pediococcus pentosaceus).
2, substratum:
MRS substratum: peptone 10g/L, extractum carnis 10g/L, yeast extract 5g/L, K
2HPO
42g/L, dibasic ammonium citrate 2g/L, sodium acetate 5g/L, glucose 20g/L, MgSO
47H
2O 0.58g/L, MnSO
4H
2O 0.19g/L, tween 80 1ml regulates pH7.0,121 ℃ of sterilization 15min.
MRS solid medium: peptone 10g/L, extractum carnis 10g/L, yeast extract 5g/L, K
2HPO
42g/L, dibasic ammonium citrate 2g/L, sodium acetate 5g/L, glucose 20g/L, MgSO
47H
2O 0.58g/L, MnSO
4H
2O 0.19g/L, tween 80 1ml regulates pH7.0, agar 10g/L.121 ℃ of sterilization 15min.
The TYG substratum: Tryptones 5g/L, yeast extract 5g/L, glucose 10g/L, sodium acetate 5g/L regulates pH7.0,121 ℃ of sterilization 15min.
The compound proliferated culture medium of potato juice: potato 200g boils 30min in 1L water, filtered through gauze removes slag.In clear liquid, add glucose 20g/L, the 10g/L peptone, the 5g/L yeast extract paste, dibasic ammonium citrate 2g/L, sodium acetate 5g/L regulates pH7.0,121 ℃ of sterilization 15min.
The compound proliferated culture medium of soybean sprout juice: soybean sprout 200g boils 30min in 1L water, filtered through gauze removes slag.In clear liquid, add the 50ml of Tomato juice's (pressing the microbiological Test preparation of milk-acid bacteria in the GB/T16347-1996. lactobacillus drink), glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L, dibasic ammonium citrate 2g/L, sodium acetate 5g/L, regulate pH7.0,121 ℃ of sterilization 15min.
The compound proliferated culture medium of wort: in 1L wort (5 degree Beaume) (" the common and commonly used fungi " of writing group with reference to Institute of Microorganism, Academia Sinica " common and commonly used fungi " is prepared), add the 50ml of Tomato juice's (pressing GB/T16347-1996 " microbiological Test of milk-acid bacteria in the lactobacillus drink " preparation), glucose 20g/L, peptone 10g/L, yeast extract paste 5g/L, dibasic ammonium citrate 2g/L, sodium acetate 5g/L regulates pH7.0,121 ℃ of sterilization 15min.
3, cell transformation method: after Pediococcus pentosaceus (Pediococcus pentosaceus) is activated, be inoculated in MRS, the compound proliferated culture medium of potato juice, substratum such as compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, cultivate 12h in 25～37 ℃, as seed liquor, inoculate MRS, the compound proliferated culture medium of potato juice, substratum such as compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice carry out enlarged culturing 24～36h, in the centrifugal collection thalline of 6000～1000r/min, then thalline is mixed with the solution or the suspension liquid of L-glutamic acid or Sodium Glutamate, in pH3.2～5.5, carry out stirring reaction 1～6d under 20～45 ℃, thereby obtain to contain the solution of γ-An Jidingsuan.
4, enzymatic conversion method method: after Pediococcus pentosaceus (Pediococcus pentosaceus) is activated, be inoculated in MRS, the compound proliferated culture medium of potato juice, substratum such as compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, cultivate 12h in 25～37 ℃, as seed liquor, inoculate MRS, the compound proliferated culture medium of potato juice, substratum such as compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice carry out enlarged culturing 24～36h, in the centrifugal collection thalline of 6000～1000r/min, then with reference to " enzyme engineering " (Guo Yong chief editor, China Light Industry Press, 2000) described method extracts the L-Glutamic decarboxylase of Pediococcus pentosaceus.Again enzyme liquid is mixed with L-glutamic acid or monosodium glutamate solution, under pH3.2～5.5,20～45 ℃, carry out stirring reaction 1h～2d, thereby obtain to contain the solution of γ-An Jidingsuan.
5, submerged fermentation method: with Pediococcus pentosaceus (Pediococcus pentosaceus) serves as to produce bacterial classification, activate earlier, seed culture medium is inserted in the back, in 25～37 ℃, 100～180rpm stir culture, 12～24h, seed liquor is inserted MRS by 4～10% inoculum sizes, the compound proliferated culture medium of potato juice, fermention mediums such as compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, 100～180rpm stirs down and cultivates 24～36h by control pH6.0～7.0, remove pH control again and continue fermentation 12～18h, the ratio of pressing the final concentration of 15～40g/L then adds L-glutamic acid or Sodium Glutamate, control pH value continues fermentation 24～96h 4.0～5.0 in 30～45 ℃, thereby acquisition contains the karusen of γ-An Jidingsuan.
Beneficial effect advantage of the present invention:
1, the present invention finds that first Pediococcus pentosaceus has very high L-Glutamic decarboxylase activity, utilize it as bacterial classification, act on L-glutamic acid or Sodium Glutamate that external source is added, not only can produce high-load GABA (maximum concentration can reach 30%), can reduce production costs greatly, and have higher security.Can be used for industries such as food, feed and medicine.
2, composition is simpler in the conversion fluid that contains GABA of cell transformation method among the present invention and enzymatic conversion method method production, is convenient to the simplification of back extraction process.The cell transformation reaction can be carried out in high density L-glutamic acid or Sodium Glutamate substrate, and the concentration of substrate height can improve usage ratio of equipment, and production concentration height, but energy efficient reduce production costs greatly.
3, the advantage of submerged fermentation method of the present invention is to adopt substep to control fermentation condition, by adding L-glutamic acid or Sodium Glutamate substrate, fermentative production GABA.This invention has solved cell proliferation and the inconsistent contradiction of tunning cumulative fermentation condition of Pediococcus pentosaceus.Zymotechnique is simple, and is easy to operate, GABA output height.
Embodiment
Embodiment 1:
After Pediococcus pentosaceus process MRS, the compound proliferated culture medium of potato juice, the compound proliferated culture medium of wort or the compound proliferated culture medium enlarged culturing of bean sprouts juice, centrifugal collection thalline, with reference to " enzyme engineering " (Guo Yong chief editor, China Light Industry Press, 2000) described method extracts the L-Glutamic decarboxylase of Pediococcus pentosaceus.Then enzyme liquid is mixed with L-glutamic acid or monosodium glutamate solution, carry out stirring reaction 1h～3d under pH3.2～5.5,20～45 ℃, can obtain concentration is 1%～20% γ-An Jidingsuan solution.
Embodiment 2:
After Pediococcus pentosaceus process MRS, the compound proliferated culture medium of potato juice, the compound proliferated culture medium of wort or the compound proliferated culture medium enlarged culturing of bean sprouts juice, centrifugal collection thalline, then thalline is mixed with L-glutamic acid or monosodium glutamate solution, in pH3.2～5.5, carry out stirring reaction 1～4d under 20～45 ℃, the centrifugation thalline can obtain concentration and be 1%～30% γ-An Jidingsuan solution.
Embodiment 3:
After Pediococcus pentosaceus process MRS, the compound proliferated culture medium of potato juice, the compound proliferated culture medium of wort or the compound proliferated culture medium enlarged culturing of bean sprouts juice, centrifugal collection thalline, then thalline is mixed with L-glutamic acid or monosodium glutamate solution, add an amount of tween (Tween), sucrose ester or Te Lidun tensio-active agents such as (Triton), in pH3.2～5.5, carry out stirring reaction 1～4d under 20～45 ℃, the centrifugation thalline can obtain concentration and be 1%～30% γ-An Jidingsuan solution.
Embodiment 4:
After Pediococcus pentosaceus process MRS, the compound proliferated culture medium of potato juice, the compound proliferated culture medium of wort or the compound proliferated culture medium enlarged culturing of bean sprouts juice, centrifugal collection thalline, adopt 0.5～10% O for toluene, centrifugal again collection thalline, then thalline is mixed with L-glutamic acid or monosodium glutamate solution, add an amount of tween (Tween), sucrose ester or Te Lidun tensio-active agents such as (Triton), in pH3.2～5.5, carry out stirring reaction 1～4d under 20～45 ℃, the centrifugation thalline can obtain concentration and be 1%～30% γ-An Jidingsuan solution.
Embodiment 5:
After Pediococcus pentosaceus process MRS, the compound proliferated culture medium of potato juice, the compound proliferated culture medium of wort or the compound proliferated culture medium enlarged culturing of bean sprouts juice, centrifugal collection thalline, adopt 0.5～10% O for toluene, centrifugal again collection thalline, then thalline is mixed with L-glutamic acid or monosodium glutamate solution, under pH3.2～5.5,20～45 ℃, carry out stirring reaction 1～4d.In the entire reaction, drip defoamer and carry out froth breaking.After reaction finished, the centrifugation thalline can obtain concentration and be 1%～30% γ-An Jidingsuan solution.
Embodiment 6:
After Pediococcus pentosaceus process MRS, the compound proliferated culture medium of potato juice, the compound proliferated culture medium of wort or the compound proliferated culture medium enlarged culturing of bean sprouts juice, centrifugal collection thalline, then thalline is mixed (semi-solid state) with the suspension liquid of L-glutamic acid or Sodium Glutamate, in pH3.2～5.5, carry out stirring reaction 1～6d under 20～45 ℃, the centrifugation thalline can obtain concentration and be 1%～30% γ-An Jidingsuan solution.
Embodiment 7:
After Pediococcus pentosaceus process MRS, the compound proliferated culture medium of potato juice, the compound proliferated culture medium of wort or the compound proliferated culture medium enlarged culturing of bean sprouts juice, centrifugal collection thalline, then thalline is mixed (semi-solid state) with the suspension liquid of L-glutamic acid or Sodium Glutamate, add an amount of tween (Tween), sucrose ester or Te Lidun tensio-active agents such as (Triton), in pH3.2～5.5, carry out stirring reaction 1～6d under 20～45 ℃, the centrifugation thalline can obtain concentration and be 1%～30% γ-An Jidingsuan solution.
Embodiment 8:
Pediococcus pentosaceus is through MRS, the compound proliferated culture medium of potato juice, after the compound proliferated culture medium enlarged culturing of compound proliferated culture medium of wort or bean sprouts juice, centrifugal collection thalline, adopt 0.5～10% O for toluene, centrifugal again collection thalline, then thalline is mixed (semi-solid state) with the suspension liquid of L-glutamic acid or Sodium Glutamate, add an amount of tween (Tween), sucrose ester or Te Lidun tensio-active agents such as (Triton), in pH3.2～5.5, carry out stirring reaction 1～6d under 20～45 ℃, the centrifugation thalline can obtain concentration and be 1%～30% γ-An Jidingsuan solution.
Embodiment 9:
After Pediococcus pentosaceus process MRS, the compound proliferated culture medium of potato juice, the compound proliferated culture medium of wort or the compound proliferated culture medium enlarged culturing of bean sprouts juice, centrifugal collection thalline, adopt 0.5～10% O for toluene, centrifugal again collection thalline, then thalline is mixed (semi-solid state) with the suspension liquid of L-glutamic acid or Sodium Glutamate, under pH3.2～5.5,20～45 ℃, carry out stirring reaction 1～6d, in the entire reaction, drip defoamer and carry out froth breaking.After reaction finished, the centrifugation thalline can obtain concentration and be 1%～30% γ-An Jidingsuan solution.
Embodiment 10:
Pediococcus pentosaceus is through MRS, the compound proliferated culture medium of potato juice, after the compound proliferated culture medium enlarged culturing of compound proliferated culture medium of wort or bean sprouts juice, centrifugal collection thalline, adopt 0.5～10% O for toluene, centrifugal again collection thalline, then thalline is carried out immobilization, L-glutamic acid or monosodium glutamate solution with pH3.2～5.5, in 20～45 ℃ of reactions, centrifugal or filtering separation immobilized cell, can obtain containing the solution of γ-An Jidingsuan, perhaps immobilized cell is adorned post, adding L-glutamic acid or monosodium glutamate solution (pH3.2～5.5) by stream, carry out continuous or intermittent reaction under 20～45 ℃, can obtain concentration is 1%～10% γ-An Jidingsuan solution.
Embodiment 11:
Pediococcus pentosaceus is through MRS, the compound proliferated culture medium of potato juice, after the compound proliferated culture medium enlarged culturing of compound proliferated culture medium of wort or bean sprouts juice, centrifugal collection thalline, then thalline is carried out immobilization, mix with L-glutamic acid or monosodium glutamate solution, under pH3.2～5.5 conditions, in 20～45 ℃ of reactions, centrifugal or filtering separation immobilized cell, can obtain containing the solution of γ-An Jidingsuan, perhaps immobilized cell is adorned post, adding L-glutamic acid or monosodium glutamate solution (pH3.2～5.5) by stream, carrying out under 30～45 ℃ continuously or intermittent reaction, can obtain concentration is 1%～10% γ-An Jidingsuan solution.
Embodiment 12:
Pediococcus pentosaceus is through MRS, the compound proliferated culture medium of potato juice, after the compound proliferated culture medium enlarged culturing of compound proliferated culture medium of wort or bean sprouts juice, centrifugal collection thalline, adopt 0.5～10% O for toluene, centrifugal again collection thalline, then thalline is carried out immobilization, mix with L-glutamic acid or monosodium glutamate solution, under pH3.2～5.5 conditions, in 20～45 ℃ of reactions, centrifugal or filtering separation immobilized cell, can obtain containing the solution of γ-An Jidingsuan, perhaps immobilized cell is adorned post, contain an amount of tween (Tween) adding by stream, the L-glutamic acid or the monosodium glutamate solution (pH3.2～5.5) of sucrose ester or Te Lidun tensio-active agents such as (Triton) carry out under 20～45 ℃ continuously or intermittent reaction, and can obtain concentration is 1%～10% γ-An Jidingsuan solution.
Embodiment 13:
After Pediococcus pentosaceus process MRS, the compound proliferated culture medium of potato juice, the compound proliferated culture medium of wort or the compound proliferated culture medium enlarged culturing of bean sprouts juice, centrifugal collection thalline, then thalline is mixed (semi-solid state) with the suspension liquid of L-glutamic acid or Sodium Glutamate, in pH3.2～5.5, carry out stirring reaction 1～6d under 20～45 ℃, when no L-glutamic acid particle exists in the reaction solution, adopt dropping HCl and NaOH solution control pH between 3.2～5.5, continue reaction 1h～2d.In the entire reaction, drip defoamer and carry out froth breaking.After reaction finished, the centrifugation thalline can obtain concentration and be 1%～30% γ-An Jidingsuan solution.
Embodiment 14:
After Pediococcus pentosaceus process MRS, the compound proliferated culture medium of potato juice, the compound proliferated culture medium of wort or the compound proliferated culture medium enlarged culturing of bean sprouts juice, centrifugal collection thalline, then thalline is mixed (semi-solid state) with the suspension liquid of L-glutamic acid or Sodium Glutamate, under pH3.2～5.5,20～45 ℃, carry out stirring reaction 1～6d.In the entire reaction, drip defoamer and carry out froth breaking.After reaction finished, the centrifugation thalline can obtain concentration and be 1%～30% γ-An Jidingsuan solution.
Embodiment 15:
After Pediococcus pentosaceus process MRS, the compound proliferated culture medium of potato juice, the compound proliferated culture medium of wort or the compound proliferated culture medium enlarged culturing of bean sprouts juice, centrifugal collection thalline, then thalline is mixed (semi-solid state) with bran acid (the L-glutamic acid crude extract of fermentative Production) suspension liquid, under pH3.2～5.5,20～45 ℃, carry out stirring reaction 1～6d.In the entire reaction, drip defoamer and carry out froth breaking.After reaction finished, the centrifugation thalline can obtain concentration and be 1%～30% γ-An Jidingsuan solution.
Embodiment 16:
Adopt the MRS inclined-plane to activate Pediococcus pentosaceus preservation bacterial classification, insert MRS, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, 100～180rpm stir culture, 12～24h, insert MRS by 4～10% then, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, 100～180rpm stirs down and adds 1mol/LNaOH control pH6.0～7.0 cultivation 24h by stream, remove pH control then, continue fermentation 12～18h, the ratio of pressing the final concentration of 15～40g/L adds L-glutamic acid or Sodium Glutamate, in 30～45 ℃, fermentation 24～96h is continued in pH4.0～5.0, is the karusen of 10～20g/L thereby obtain alpha-aminobutyric acid content.
Embodiment 17:
With the Pediococcus pentosaceus is to produce bacterial classification, adopt the MRS inclined-plane to activate earlier, again with triangular flask MRS liquid culture based on 25～37 ℃, the 100rpm shaking table is cultivated 12h and is activated, press 4～10% inoculum size access MRS then, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, 100～180rpm stir culture, 12～24h, insert MRS by 4～10% then, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, 100～180rpm stirs down and adds 1mol/LNaOH control pH6.0～7.0 cultivation 24h by stream, remove pH control then, continue fermentation 12～18h, the ratio of pressing the final concentration of 15～40g/L adds L-glutamic acid or Sodium Glutamate, add NaOH and HCl control pH value by stream and continue fermentation 24～96h in 30～45 ℃, thereby the acquisition alpha-aminobutyric acid content is the karusen of 10～20g/L 4.0～5.0.Through the sterilization discharging, can carry out GABA and extract and make with extra care.
Embodiment 18:
With the Pediococcus pentosaceus is to produce bacterial classification, adopt the MRS inclined-plane to activate earlier, again with triangular flask MRS liquid culture based on 25～37 ℃, the 100rpm shaking table is cultivated 12h and is activated, press 4～10% inoculum size access MRS then, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, 100～180rpm stir culture, 12～24h, insert MRS by 4～10% then, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, 100～180rpm stirs down and adds 1mol/LNaOH control pH6.0～7.0 cultivations, 24～36h by stream, the ratio of pressing the final concentration of 15～40g/L adds L-glutamic acid or Sodium Glutamate, add NaOH and HCl control pH value by stream and continue fermentation 24～96h in 30～45 ℃, thereby the acquisition alpha-aminobutyric acid content is the karusen of 10～20g/L 4.0～5.0.
Embodiment 19:
With the Pediococcus pentosaceus is to produce bacterial classification, adopt the MRS inclined-plane to activate earlier, again with triangular flask MRS liquid culture based on 25～37 ℃, the 100rpm shaking table is cultivated 12～24h and is activated, press 4～10% inoculum size access MRS then, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, 100～180rpm stir culture 12h, insert MRS by 4～10% then, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, 100～180rpm stirs down and adds 1mol/LNaOH control pH6.0～7.0 cultivation 24h by stream, remove pH control then, continue fermentation 12～18h, the ratio of pressing the final concentration of 15～40g/L adds L-glutamic acid or Sodium Glutamate, add NaOH and HCl control pH value by stream and continue fermentation 24～96h in 30～45 ℃, thereby the acquisition alpha-aminobutyric acid content is the karusen of 10～20g/L 4.0～5.0.Through the sterilization discharging, can carry out GABA and extract and make with extra care.
Embodiment 20:
With the Pediococcus pentosaceus is to produce bacterial classification, adopt the MRS inclined-plane to activate earlier, again with triangular flask MRS liquid culture based on 25～37 ℃, the 100rpm shaking table is cultivated 12～24h and is activated, press 4～10% inoculum size access MRS then, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, 100～180rpm stir culture, 12～24h, insert MRS by 4～10% then, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, pH6.0～7.0,100～180rpm stirs and cultivates 24h down, remove pH control then, continue fermentation 12～18h, ratio by the final concentration of 15～40g/L adds L-glutamic acid or Sodium Glutamate, control pH value then and continue fermentation 24～96h 4.0～5.0 in 30～45 ℃, thereby the acquisition alpha-aminobutyric acid content is the karusen of 10～20g/L.Through the sterilization discharging, can carry out GABA and extract and make with extra care.
Embodiment 21:
With the Pediococcus pentosaceus is to produce bacterial classification, adopt the MRS inclined-plane to activate earlier, insert MRS, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, pH6.0～7.0,100～180rpm stir culture 12h, insert MRS by 4～10% then, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, pH6.0～7.0,100～180rpm stirs and cultivates 24h down, remove pH control then, continue fermentation 12～18h, the ratio of pressing the final concentration of 15～40g/L adds L-glutamic acid or Sodium Glutamate, controlling the pH value then and continue fermentation 24～96h 4.0～5.0 in 30～45 ℃, is the karusen of 10～20g/L thereby obtain alpha-aminobutyric acid content.Through the sterilization discharging, can carry out GABA and extract and make with extra care.
Claims (1)
1. method with Pediococcus pentosaceus (Pediococcus pentosaceus) biosynthesizing γ-An Jidingsuan, it is characterized by: as producing bacterial classification, produce the method for γ-An Jidingsuan by cell transformation method, enzymatic conversion method method or tank fermentation method with Pediococcus pentosaceus (Pediococcus pentosaceus); Being characterized as of cell transformation method: after Pediococcus pentosaceus (Pediococcus pentosaceus) is activated, be inoculated in MRS, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, cultivate 12h in 25～37 ℃, as seed liquor, inoculate MRS, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice carry out enlarged culturing 24～36h, in the centrifugal collection thalline of 6000～1000r/min, then thalline is mixed with the solution or the suspension liquid of L-glutamic acid or Sodium Glutamate, in pH3.2～5.5, carry out stirring reaction 1～6d under 20～45 ℃, thereby obtain to contain the solution of γ-An Jidingsuan; Being characterized as of enzymatic conversion method method: after Pediococcus pentosaceus (Pediococcus pentosaceus) is activated, be inoculated in MRS, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, cultivate 12h in 25～37 ℃, as seed liquor, inoculate MRS, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice carry out enlarged culturing 24～36h, in the centrifugal collection thalline of 6000～1000r/min, L-Glutamic decarboxylase to Pediococcus pentosaceus extracts then, again enzyme liquid is mixed with L-glutamic acid or monosodium glutamate solution, in pH3.2～5.5, carry out stirring reaction 1h～2d under 20～45 ℃, thereby obtain to contain the solution of γ-An Jidingsuan; Being characterized as of tank fermentation method: with Pediococcus pentosaceus (Pediococcus pentosaceus) serves as to produce bacterial classification, activate earlier, MRS is inserted in the back, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, 100～180rpm stir culture, 12～24h, as seed liquor, seed liquor is inserted MRS by 4～10% inoculum sizes, the compound proliferated culture medium of potato juice, compound proliferated culture medium of wort or the compound proliferated culture medium of bean sprouts juice, in 25～37 ℃, 100～180rpm stirs down and cultivates 24～36h by control pH6.0～7.0, remove pH control again and continue fermentation 12～18h, the ratio of pressing the final concentration of 15～40g/L then adds L-glutamic acid or Sodium Glutamate, control pH value continues fermentation 24～96h 4.0～5.0 in 30～45 ℃, thereby acquisition contains the karusen of γ-An Jidingsuan.
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| 李云 等.产谷氨酸脱羧酶片球菌的鉴定及其酶学性质.《食品科学》.2010,第31卷(第9期),第187-191页. * |
| 李云 等.利用戊糖片球菌HS2制备γ-氨基丁酸.《"亚运食品安全与广东食品产业创新发展"学术研讨会暨2009年广东省食品学会年会论文集》.2009,第89-94页. |
| 李云 等.戊糖片球菌HS2细胞制备γ-氨基丁酸的研究.《湖北农业科学》.2010,第49卷(第6期),第1450-1453页. * |
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