CN1718735A - Utilize the bacillus natto solid fermentation to produce polyglutamic acid and product application - Google Patents
Utilize the bacillus natto solid fermentation to produce polyglutamic acid and product application Download PDFInfo
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- CN1718735A CN1718735A CN 200410043690 CN200410043690A CN1718735A CN 1718735 A CN1718735 A CN 1718735A CN 200410043690 CN200410043690 CN 200410043690 CN 200410043690 A CN200410043690 A CN 200410043690A CN 1718735 A CN1718735 A CN 1718735A
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Abstract
Utilize the bacillus natto solid fermentation to produce polyglutamic acid and product application.The production method of the γ-polyglutamic acid of research is solution fermentation at present, does not find to adopt soy bean solid fermentation process of the present invention as yet.Method of the present invention comprises: with Soybean Pretreatment, the inoculation bacillus natto, it is characterized in that: make bacillus natto be evenly distributed in the surface of soybean by stirring, constant-temperature moisture-keeping 1-2 stirs the γ-polyglutamic acid of extraction bacillus natto secretion on the soybean surface with physiological saline round the clock, the centrifugation thalline, supernatant liquor adds original volume 1-2 ethanol doubly, behind the diel through the ultrafiltration of 300000D membrane stack, the centrifugation thalline obtains γ-polyglutamic acid crude product and vitamin K respectively
2Mixing solutions and Nattokinase genus bacillus.γ-polyglutamic acid is the bioabsorbable polymer material of a kind of All Pure Nature, multifunctionality, Biodegradable, in medicine, chemical industry, foodstuffs industry, environment, agricultural, forestry and other field extensive use is arranged.
Description
Technical field: the present invention relates to a kind of fermentation engineering of technical field of bioengineering, the new and high technology in the enzyme engineering, the solid fermentation that promptly utilizes bacillus natto to pass through soybean is produced the method for γ-polyglutamic acid and the comprehensive utilization of products obtained therefrom thereof.
Background technology: the production method of the γ-polyglutamic acid of research is solution fermentation at present, does not find to adopt soy bean solid fermentation process of the present invention as yet.
Summary of the invention: the purpose of this invention is to provide and a kind ofly utilize soybean to make substratum, the inoculation bacillus natto is produced the integrated application of the method and products thereof of γ-polyglutamic acid by solid fermentation.
Above-mentioned purpose realizes by following technical scheme:
Utilize the bacillus natto solid fermentation to produce the polyglutamic acid method, its composition comprises: with Soybean Pretreatment, the inoculation bacillus natto, make bacillus natto be evenly distributed in the surface of soybean by stirring, constant-temperature moisture-keeping 1-2 round the clock, stir the γ-polyglutamic acid of extraction bacillus natto secretion with physiological saline on the soybean surface, the centrifugation thalline, supernatant liquor is through the ultrafiltration of 300000D membrane stack, add original volume 1-2 ethanol doubly, behind the diel, the centrifugation thalline obtains γ-polyglutamic acid crude product and vitamin K respectively
2Mixing solutions and Nattokinase genus bacillus.
The described bacillus natto solid fermentation that utilizes is produced the polyglutamic acid method, the crude product of obtaining is purified, its process comprises: with resulting γ-polyglutamic acid crude product water dissolution, add 1-1.5 times of volume of ethanol, centrifugation after 24 hours, with after the washing with alcohol, vacuum is drained and is obtained γ-polyglutamic acid elaboration once more.
The described bacillus natto solid fermentation that utilizes is produced the polyglutamic acid method, describedly soybean is carried out pre-treatment comprise: described soybean flowing water is soaked diel, drench moisture, put into autoclave, 130 ℃, autoclaving 1-2 hour, after the taking-up, put in the sterilisable chamber, to be cooled to 50 ℃, inoculate.
The described bacillus natto solid fermentation that utilizes is produced the polyglutamic acid method, and the part by weight of described inoculation is 2% of soybean parts by weight, changes with per second 190 at shaking table then, cultivates 1-2 round the clock, and bacterium number/ml should be 1 * 10
8Individual/ml, as bacterial classification, press 10%V/W, be inoculated in 50 ℃ the soybean, fully stir, make bacterial classification be uniformly distributed in the soybean surface, postvaccinal culturing room keeps 39-40 ℃ constant temperature, the indoor humidity strictness remains on more than 95%, in case fermented soybean surface drying and influence the output of γ-polyglutamic acid, fermentation 2 round the clock.
The described bacillus natto solid fermentation that utilizes is produced the polyglutamic acid method, describedly stir the bacillus natto secretion with normal saline extraction and comprise: stir with 0.9% NaCl of 5-10 times of volume of fermented soybean and extracted 2 hours in the process of the γ-polyglutamic acid on soybean surface, after 100 eye mesh screens filter, collect filtrate, filtrate is continuously centrifuged under 10000 rev/mins of situations again, obtains supernatant liquor (thick).Throw out can be used to reclaim thalline, makes the bacillus natto live bacteria agent after the freeze-drying, utilizes the high pressure liquid chromatography (HPLC) method to measure the concentration of γ-polyglutamic acid simultaneously.
The described bacillus natto solid fermentation that utilizes is produced the polyglutamic acid method, and after supermembrane filtered, collection was held back with ultrafiltration and gone out liquid two portions respectively.Ultrafiltration is held back when γ-polyglutamic acid concentration is for 40-60g/L in the part to be ended, then, to wherein adding 1-2 times of volume of ethanol, stir, 4 ℃, put 24 hours, precipitation γ-polyglutamic acid, 6000 rev/mins centrifugal 30 minutes, reclaim γ-polyglutamic acid and obtain γ-polyglutamic acid crude product, exceed liquid and extract Nattokinase and vitamin K in order to reclaim
2
The described bacillus natto solid fermentation that utilizes is produced the polyglutamic acid method, it is characterized in that: the concrete grammar that the crude product of obtaining is withdrawn deposit is: with the γ-polyglutamic acid raw product that is obtained, use dissolved in distilled water, making its γ-polyglutamic acid concentration is 60g/L, after removing insolubles, again to wherein adding 1-1.5 times of volume of ethanol at 4 ℃, left standstill 24 hours, centrifugal 30 minutes at 6000 rev/mins, collect γ-polyglutamic acid, then with 1-2 absolute ethanol washing doubly, dehydration, vacuum is drained, and obtains the pure product of γ-polyglutamic acid of purifying.
To extract vitamin K
2Raw material as the preventing osteoporosis disease drug.
With the sedimentary Nattokinase of centrifugal recovery, as the thrombolysis capsule raw material.
With after the fermented soybean homogenate as producing soybean protein peptide, the raw material of soybean step-down peptide.
This technical scheme has following beneficial effect:
What 1, use filtered out is high yield γ-polyglutamic acid, and as bacterial classification, producing soybean with Heilungkiang is fermentation raw material with the bacillus natto bacterial strain Bacillus subtilis natto HW-1 that produces Nattokinase.Obtain γ-polyglutamic acid by solid fermentating mode, opened up cheap, new production route efficiently for the production of polyglutamic acid, the comprehensive development and utilization to soybean has played good promoter action simultaneously.
2. present method is at the process for separating and purifying with twice ethanol sedimentation, can obtain purifying γ-polyglutamic acid (be the product of γ-PGA), yield 10-20%, purity is more than 95%.
3. the present invention is a kind of fermentation engineering of using, the technological method of mass production novel high polymer material.But utilize not only mass production γ-PGA of the present invention, can produce multiple useful valuable product simultaneously, can create tangible economy and society benefit.Characteristics of the present invention are: produce several products of supporting utilization in a kind of bacteria solid fermentation, comprising r-PGA, NK, VK
2, freeze-dried, the soybean protein of Bisubtilis notto viable bacteria and soybean protein peptide etc.
4. product of the present invention: γ-polyglutamic acid is the biopolymer product of a kind of All Pure Nature, multifunctionality, Biodegradable.Molecular weight can be applicable to various fields between 500000D-2000000D.Be applied to pharmaceutically, new drug carrier, gets promotor etc. at medicine buffer reagent, solubility promoter.Biological medical polymer material, biodegradable, as polyglutamic acid taxol carcinostatic agent, improve curative effect and utilization ratio, suture material, artificial skin etc.On the agricultural, the sand-fixing vegetation novel material, bag, can be guaranteed seed germination, emerge in the water deficient regions, desert by plant seed, prevents the influence of arid, and water conservation reaches 1: 3500 times.New liquid fertilizer, the slow release and the loss that keep fertility.Industry and other field, water and wastewater treatment, as sequestrant, sorbent material, flocculation agent dispose of sewage, heavy metal, radio active material, makeup, foodstuffs industry; Filter membrane material, material which can retain moisture, wrapping material, tissue engineering material etc.
5. simultaneously, from extracting solution, take into account production Nattokinase and vitamin K
2, the fermentation natto continues to utilize preparation soybean protein or soybean protein peptide, and therefore a kind of soy bean solid fermentation can be produced multiple product, almost completely is utilized, and waste is seldom.So can create maximum value, if produce then single product of γ-PGA with solution fermentation, raw materials consumption is bigger, and cost is higher.
Description of drawings: accompanying drawing 1 is the process and the comprehensive utilization synoptic diagram of present method.
Accompanying drawing 2 is HPLC analysis charts of soy bean solid fermentative production γ-PGA, and 1 is that standard substance γ-PGA, 2 is that crude product γ-PGA, 3 is the γ-PGA with the inventive method purifying.
Accompanying drawing 3 is SDS-polyacrylamide gel electrophoresis figure of γ-PGA, and wherein 1-7 is a sample, and M is a molecular weight
The specific embodiment of the present invention:
Embodiment 1:
The activation and the cultivation of bacillus natto (Bacillus subtilis natto HW-1).The Bacillus subtilis natto HW-1 bacterial classification of going bail for and depositing, streak culture on the LB plate, 18h, after the taking-up, choose single bacterium colony from plate and be connected to the 5mlLB/ pipe 37 ℃, 180rpm, 18 hours,, insert in the 100mlLB/500ml triangular flask again by 2% inoculum size, 37 ℃, cultivated 18 hours, as the solid fermentation bacterial classification, standby.
Select soybean, discard broken incomplete soybean, soaked 18 hours under flowing water then, control removes moisture, divides to install in the dish, and 130 ℃, autoclaving 1 hour after the taking-up, is put transfer room, waits to inoculate.
After sterilization, when temperature is reduced to 50 ℃ of left and right sides,, bacterial classification is linked in the soybean dish by 10% (V/W), fully stir, the soybean surface is inoculated equably, will present this moment does not promptly have redundant solution in the dish, and the soybean surface is moist again to be good.
The soybean dish that inoculation is good is multiple puts into culturing room with sterile gauze, cultivates 48 hours for 39-40 ℃, and indoor humidity is greater than 95%, and is placed with the basin of several dress sterilized waters, to guarantee indoor humidity, prevents the soybean surface drying, influences bacteria growing and γ-PGA output.
Cultivate after 48 hours, the soybean surface presents one deck adhesive material, can pull out very long silk, merges fermented soybean, and with the physiological saline of 5-10 times of volume, washing and stirring were extracted 2 hours, and 100 eye mesh screens filter, and collect filtrate and fermented soybean respectively.
Filtrate, at 10000rpm, continuously centrifuged is collected centrifugal liquid, is r-PGA and Nattokinase extracting solution.
Extracting solution in last, the filter ultrafiltration of using 300000D makes γ in the trapped fluid-PGA concentration when 40-60g/L with concentrated, stops ultrafiltration, and this moment, the trapped fluid viscosity was very big.
In concentrated solution, add 1-1.5 times of ethanol, fully stir, put 4 ℃, spend the night, be settled out γ-PGA, centrifugal 6000rpm, 30 minutes, its throw out was the raw product of γ-PGA.
Weigh and go up the γ-PGA crude product of item, it is 60g/L that dissolving makes its concentration, once more with 1-1.5 times of ethanol sedimentation γ-PGA, 4 ℃, spend the night, centrifugal 6000rpm, 30min gets throw out, with the dehydrated alcohol dehydration of 1-2 times of volume, vacuum is drained, and weighs, and obtains γ-PGA purified product.
Above-mentioned 300000D filter membrane exceeds solution, uses the 5000D film, and ultrafiltration and concentration, concentrated solution be with 2 times of ethanol sedimentations, centrifugal 6000rpm, and 30 minutes, reclaim Nattokinase, be the Nattokinase raw material, do not contain vitamin K in this enzyme
2
Exceed liquid at the 5000D film, concentrating under reduced pressure 1/10 volume is used organic ethanol-extracted, reclaims VK
2
Fermented soybean, adding distil water, homogenate, precipitate soybean protein.
Soy bean proteinous soln obtains soybean protein peptide with neutral and acid protease hydrolysis spraying drying.
Embodiment 2:
One, the technical essential of present embodiment
That use filters out is high yield γ-PGA, and the bacterial strain Bacillus subtilis nattoHW-1 that produces MK is arranged.Producing soybean with Heilungkiang is fermentation raw material.Twice ethanol sedimentation separates purification γ-PGA.
Two, experimental implementation step (see figure 1)
1, culture of strains
Preserve bacterial classification No. 1 and be linked into (5mlLB/ props up) in the actication of culture pipe, 37 ℃, shaking table is cultivated, 190rpm, and 18 hours, be linked in the 50ml LB/250ml triangular flask by 2% inoculum size then, 37 ℃, shaking table shaking culture, 200rpm, 18 hours.As bacterial classification, standby.
2, soybean 1000g, flowing water soaked 24 hours, were filled in the dish, and 130 ℃ of sterilizations 1 hour are put after the taking-up and connect the bacterium chamber, are chilled to 50-60 ℃, get bacterial strain No. 1, were seeded in the dish by 10%, and abundant mixing coated with sterile gauze, is put in the incubator 39 ℃-40 ℃ and cultivated 2 days.Keep humidity greater than 95%, in case the soybean surface drying in the incubator.
3, extract purifying: after the soy bean solid fermentation, produce a large amount of viscous liquids on the surface, mainly contain γ-PGA, the Nattokinase that fermentation simultaneously produces comprises wherein, with the physiological saline of 5-10 times of volume, stir and extract γ-PGA, 2-4 hour, divide 2-3 time, united extraction liquid then, centrifugal 30 minutes of 10000rpm collects supernatant liquor, concentrate through 300000 ultra-filtration membranes again, after being concentrated into 40-60g/L, add the cold ethanol precipitation γ-PGA of 1-2 times of volume, refrigerator overnight, centrifugal, 6000rmp, 30 minutes, collecting precipitation, after the dehydrated alcohol dehydration, drain and obtain raw product γ-PGA.
Embodiment 3:
For the γ-PGA that purifies, be dissolved in the γ-PGA of ethanol sedimentation in the physiological saline again, make the centrifugal insolubles of removing of concentration 40-60g/L, add 1-2 times of volume ethanol again, the refrigerator precipitation is spent the night the centrifugal γ-PGA that obtains, the dehydrated alcohol dehydration, drain and obtain γ-PGA, purity＞95%, productive rate is the 80-120g/Kg soybean.
The dried soybean of 1000g, soaking the back is 2200g, extracts purifying through solid fermentation, gets 115g crude product γ-PGA, is further purified the γ-PGA that obtains the 89.5g purifying, the γ behind the purifying-PGA state is a white powder.
Embodiment 4.
One, the technical essential of this experimental example:
1, uses the content of high-pressure liquid phase chromatogram therapy determining γ-PGA; 2, adopt HCL hydrolysis γ-PGA, be L-glutamic acid, or directly use γ-PGA,, detect at the 360nm wavelength with amino acid column front derivation method.
Two, test operation step
1, adopt amino acid column front derivation method to measure γ-PGA content, the HCL hydrolysis of concrete grammar: γ-PGA accurately takes by weighing 10mg γ-PGA dry sample, melt into 1ml adds equal-volume 12NHCL, 105 ℃, hydrolysis 8h adds NaOH and transfers to pH7.0, is settled to 100ml then.
2, derive: the γ that water intaking is separated-PGA1ml adds in the dissolve measuring bottle, adds 0.5M/L NaHCO respectively
3(pH9.0) 1ml and 1% 2, the acetonitrile solution 1ml of 4-dinitrofluorobenzene derivating agent, after being mixed, with 60 ℃ of down reactions 1 hour, be cooled to room temperature after, add 0.01M/L KH
2PO
4(pH7.0), and be settled to 10ml, get a certain amount of 0.45um of using membrane filtration, get 10ul and carry out the HPLC analysis, sample should keep in Dark Place at 4 ℃.
3, chromatographiccondition and method: mobile phase A: acetonitrile+water (1: 1): B:60mM/L NaAc (pH6.4) contains 10%N, dinethylformamide; 35 ℃ of column temperatures, flow velocity 0.5ml/min.Detector: ultraviolet 360nm; Gradient elution: time (min) 0,4,32,50,56, B (%) 84,64,40,8,74.Mix up baseline, put sample well, wash post, sample introduction detects.
4, the γ-PGA of crude product and purifying, HPLC measures as Fig. 2 finding, and its result lists in the table 1.
The HPLC analytical data of table 1, γ-PGA sample
| Sample number | Peak area | Peak heights | Concentration % | Output g/kg |
| Standard substance raw product purifying product | 10359394 14228396 17302432 | 245952 298347 589287 | 93.97 80.47 95.33 | ------ 115 89.5 |
Listing in the table 1 with L-glutamic acid is standard substance, and HCL hydrolyzation sample HPLC measures relevant data, comparison shows that the γ-PGA product of No. 1 bacterial strain from data, and after purifying through the secondary ethanol sedimentation, quality is good, has only small amount of impurities.Thereby show: measure r-PGA in fermented liquid and to extract samples contg be feasible with dinitrofluorobenzene column front derivation method HPLC, method is accurate, and is reliable, easily row.
Embodiment 5:
One, the technical essential of this test example:
1, the sample glue and the separation gel that accurately prepare 10% polyacrylamide guarantee that molecular weight of albumen standard and γ-PGA can fine the separation, show clear band.
2, handle sample with SDS.
Two, experimental implementation and step:
1, prepare SDS-polyacrylamide gel method routinely, make 10% separation gel, treat its polymerization after, be prepared into sample glue thereon, behind the sample gummed, in the electrophoresis chamber of packing into.
2, quantitatively take by weighing γ-PGA sample, be dissolved into the sample of concentration 1-10 μ g/ μ l with the IBS damping fluid.
3, get 5 μ l samples and add 5 μ l sample loading buffers, boiled 3-5 minute in 100 ℃ of boiling water, after the cooling, moment is centrifugal, and getting supernatant liquor is the electrophoresis sample, and standard molecular weight albumen is operated equally.
4, beginning electrophoresis, voltage is at 8mV/cm during beginning, and after sample entered separation gel, accent voltage was 15mV/cm, and electrophoresis to tetrabromophenol sulfonphthalein arrives the glue lower end.
5, take out running gel, gel with water rinse after, dyeing is 3-4 hour in Coomassie brilliant blue, main development criteria albumen, γ-PGA then is not colored, dyeing stops, and decolours three times with methyl alcohol-HAc, obtains albumen colour developing gel, dyeed 3-4 hour at methylene blue solution then with 0.5%, taking-up rinses out dyestuff, decolours to the glue body when very shallow, can find clear high molecular γ-PGA band, take a picture as Fig. 3, visible r-PGA is a high-molecular weight compounds on Fig. 3, on 10% gel, finds no other protein staining band.
Claims (10)
1. one kind is utilized the bacillus natto solid fermentation to produce the polyglutamic acid method, its composition comprises: with Soybean Pretreatment, the inoculation bacillus natto, it is characterized in that: make bacillus natto be evenly distributed in the surface of soybean by stirring, constant-temperature moisture-keeping 1-2 round the clock, stir the γ-polyglutamic acid of extraction bacillus natto secretion with physiological saline on the soybean surface, the centrifugation thalline, supernatant liquor is through the ultrafiltration of 300000D membrane stack, add original volume 1-2 ethanol doubly, behind the diel, the centrifugation thalline obtains γ-polyglutamic acid crude product and vitamin K respectively
2Mixing solutions and Nattokinase genus bacillus.
2. the bacillus natto solid fermentation that utilizes according to claim 1 is produced the polyglutamic acid method, it is characterized in that: the crude product of obtaining is purified, its process comprises: with resulting γ-polyglutamic acid crude product water dissolution, add 1-1.5 times of volume of ethanol, centrifugation after 24 hours, with after the washing with alcohol, vacuum is drained and is obtained γ-polyglutamic acid elaboration once more.
3. produce the polyglutamic acid method according to claim 1 or the 2 described bacillus natto solid fermentations that utilize, it is characterized in that: describedly soybean is carried out pre-treatment comprise: described soybean flowing water is soaked diel, drench moisture, put into autoclave, 130 ℃, autoclaving 1-2 hour, after the taking-up, put in the sterilisable chamber, to be cooled to 50 ℃, inoculate.
4. the bacillus natto solid fermentation that utilizes according to claim 1 and 2 is produced the polyglutamic acid method, it is characterized in that: the part by weight of described inoculation is 2% of soybean parts by weight, change with per second 190 at shaking table then, cultivate 1-2 round the clock, bacterium number/ml should be 1 * 10
8Individual/ml, as bacterial classification, press 10%V/W, be inoculated in 50 ℃ the soybean, fully stir, make bacterial classification be uniformly distributed in the soybean surface, postvaccinal culturing room keeps 39-40 ℃ constant temperature, the indoor humidity strictness remains on more than 95%, in case fermented soybean surface drying and influence the output of γ-polyglutamic acid, fermentation 2 round the clock.
According to claim 12 or the described bacillus natto solid fermentation that utilizes produce the polyglutamic acid method, it is characterized in that: describedly stir the bacillus natto secretion with normal saline extraction and comprise: stir with 0.9% NaCl of 5-10 times of volume of fermented soybean and extracted 2 hours in the process of the γ-polyglutamic acid on soybean surface, after 100 eye mesh screens filter, collect filtrate, filtrate is continuously centrifuged under 10000 rev/mins of situations again, obtains supernatant liquor (thick).Throw out can be used to reclaim thalline, makes the bacillus natto live bacteria agent after the freeze-drying, utilizes the high pressure liquid chromatography (HPLC) method to measure the concentration of γ-polyglutamic acid simultaneously.
6. the bacillus natto solid fermentation that utilizes according to claim 1 and 2 is produced the polyglutamic acid method, it is characterized in that: after supermembrane filtered, collection was held back with ultrafiltration and is gone out liquid two portions respectively.Ultrafiltration is held back when γ-polyglutamic acid concentration is for 40-60g/L in the part to be ended, then, to wherein adding 1-2 times of volume of ethanol, stir, 4 ℃, put 24 hours, precipitation γ-polyglutamic acid, 6000 rev/mins centrifugal 30 minutes, reclaim γ-polyglutamic acid and obtain γ-polyglutamic acid crude product, exceed liquid and extract Nattokinase and vitamin K in order to reclaim
2
7. the bacillus natto solid fermentation that utilizes according to claim 1 and 2 is produced the polyglutamic acid method, it is characterized in that: the concrete grammar that the crude product of obtaining is withdrawn deposit is: with the γ-polyglutamic acid raw product that is obtained, use dissolved in distilled water, making its γ-polyglutamic acid concentration is 60g/L, again to wherein adding 1-1.5 times of volume of ethanol at 4 ℃, left standstill 24 hours, centrifugal 30 minutes at 6000 rev/mins, collect γ-polyglutamic acid, then with 1-2 absolute ethanol washing doubly, dehydration, vacuum is drained, and obtains the pure product of γ-polyglutamic acid of purifying.
8. will extract vitamin K
2Raw material as the preventing osteoporosis disease drug.
9. with the sedimentary Nattokinase of centrifugal recovery, as the thrombolysis capsule raw material.
With after the fermented soybean homogenate as producing soybean protein peptide, the raw material of soybean step-down peptide.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1943411B (en) * | 2006-10-26 | 2010-05-26 | 中国农业科学院油料作物研究所 | High efficiency continuously producing method for natto functional active components |
| CN101948769A (en) * | 2010-08-20 | 2011-01-19 | 南京农业大学 | Bacteria YXY-C1 for preparing gamma-polyglutamic acid by solid fermentation and products thereof |
| CN102533885A (en) * | 2012-01-09 | 2012-07-04 | 东莞理工学院 | Method for producing gamma-polyglutamic acid through adding sodium chloride (NaCl) in fermentation process |
| CN102649972A (en) * | 2011-02-23 | 2012-08-29 | 浙江长三角生物技术股份有限公司 | Method for performing solid state fermentation on natto gum |
| CN103039966A (en) * | 2011-10-14 | 2013-04-17 | 沈阳科健生物技术有限公司 | Method for separating thrombolytic substances from solid state fermented natto |
| CN105316370A (en) * | 2014-07-28 | 2016-02-10 | 中国海洋大学 | Method for producing gamma-polyglutamic acid by using solid-state fermentation of Bacillus natto |
| CN105769667A (en) * | 2016-03-18 | 2016-07-20 | 无限极（中国）有限公司 | Lucid ganoderma fermentation liquor preparation method and application of lucid ganoderma fermentation liquor |
| CN110117625A (en) * | 2019-05-15 | 2019-08-13 | 烟台东方蛋白科技有限公司 | A kind of technique using glass noodle processing technique waste water fermenting and producing polyglutamic acid |
| CN111205921A (en) * | 2020-03-11 | 2020-05-29 | 山东惠仕莱生物科技有限公司 | Preparation method of edible oil rich in natural MK-7 |
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| CN1346891A (en) * | 2001-09-29 | 2002-05-01 | 南京工业大学 | Process for prepering gamma-polyglutamic acid and polyglutamates |
| CN1164737C (en) * | 2002-12-30 | 2004-09-01 | 南京工业大学 | Preparing gamma-poly glutamic acid, glutamate, glutathione and its precursor with bacillus subtilis NX-2 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1943411B (en) * | 2006-10-26 | 2010-05-26 | 中国农业科学院油料作物研究所 | High efficiency continuously producing method for natto functional active components |
| CN101948769A (en) * | 2010-08-20 | 2011-01-19 | 南京农业大学 | Bacteria YXY-C1 for preparing gamma-polyglutamic acid by solid fermentation and products thereof |
| CN102649972A (en) * | 2011-02-23 | 2012-08-29 | 浙江长三角生物技术股份有限公司 | Method for performing solid state fermentation on natto gum |
| CN103039966A (en) * | 2011-10-14 | 2013-04-17 | 沈阳科健生物技术有限公司 | Method for separating thrombolytic substances from solid state fermented natto |
| CN102533885A (en) * | 2012-01-09 | 2012-07-04 | 东莞理工学院 | Method for producing gamma-polyglutamic acid through adding sodium chloride (NaCl) in fermentation process |
| CN102533885B (en) * | 2012-01-09 | 2015-02-04 | 东莞理工学院 | Method for producing gamma-polyglutamic acid through adding sodium chloride (NaCl) in fermentation process |
| CN105316370A (en) * | 2014-07-28 | 2016-02-10 | 中国海洋大学 | Method for producing gamma-polyglutamic acid by using solid-state fermentation of Bacillus natto |
| CN105769667A (en) * | 2016-03-18 | 2016-07-20 | 无限极（中国）有限公司 | Lucid ganoderma fermentation liquor preparation method and application of lucid ganoderma fermentation liquor |
| CN110117625A (en) * | 2019-05-15 | 2019-08-13 | 烟台东方蛋白科技有限公司 | A kind of technique using glass noodle processing technique waste water fermenting and producing polyglutamic acid |
| CN111205921A (en) * | 2020-03-11 | 2020-05-29 | 山东惠仕莱生物科技有限公司 | Preparation method of edible oil rich in natural MK-7 |
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