CN109136214A - A kind of preparation method and application of fixing lactic acid bacteria - Google Patents
A kind of preparation method and application of fixing lactic acid bacteria Download PDFInfo
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Abstract
The invention discloses a kind of preparation method and application of fixing lactic acid bacteria, which includes the following steps: S1, making lactic acid bacterium concentrate: the lactic acid bacteria suspension centrifugal concentrating after enrichment culture is obtained lactic acid bacterium concentrate；S2, lactic acid bacteria are fixed: 1) with water-swellable sericin, obtaining sericin solution, sericin solution is uniformly mixed with lactic acid bacterium concentrate, obtains liquid A；2) liquid A obtained to the step 1) instills crosslinking agent, obtains the sericin hydrogel containing lactic acid bacteria；3) cellulose powder is dissolved in the cellulose solution that water is swollen, and the cellulose solution through being swollen is added into the sericin hydrogel that the step 2) obtains, and mixes post-crosslinking；4) crosslinked fluid that step 3) obtains slowly is instilled into calcium chloride solution by constant flow pump, then filtered, deionized water washing obtains fixing lactic acid bacteria, and 4 DEG C of refrigerators save.The lactic acid bacteria of immobilization of the present invention is for preparation γ-aminobutyric acid of fermenting.
Description
Technical field
The invention belongs to fixing lactic acid bacteria preparation technical fields, and in particular to a kind of preparation method of fixing lactic acid bacteria
And application.
Background technique
γ-aminobutyric acid (GABA) also known as amino acid injection-800, are a kind of white crystalline powders, are distributed widely in animal and plant body
Interior non-protein amino acid is the main inhibitory neurotransmitter of central nervous system.It has an ammonia on the position γ of butyric acid
Base is highly soluble in water, is slightly soluble in hot ethanol, be not readily dissolved in alcohol, ether and phenol with the presence of non-binding state, is had and is similar to glutamic acid
Sweet tea hide, the flavor of food can be enhanced.Its molecular formula is C4H9NO2, molecular weight 103.12.
GABA can inhibit central nervous system and be overexcited, and have stable effect to brain, and then promote to loosen and eliminate
It is nervous, there is important physiological function: (1) improving memory, enhance memory function；(2) to the rush of growth hormone secretion
The effects of into effect, hormone of growing up has the enhancing for promoting skeletal muscle, improves immunity, and reduction is fatty, improves body function；
(3) analgesia, calm and improvement sleep effect；(4) there are alleviation and therapeutic effect to hypertension.
γ-aminobutyric acid preparation method mainly has chemical synthesis and two kinds of biological synthesis process, and chemical synthesis process is main
It is (180 DEG C) reactions under intense conditions with O-phthalic imide potassium and γ-neoprene cyanogen, product and concentrated sulfuric acid back hydrolysis,
It is obtained using crystallization and purification.Although chemical synthesis GABA is swift in response, but violent, retrial is by force with reaction condition,
The disadvantages of energy consumption is high, at high cost, side reaction is more and environmental pollution is serious.In comparison biological synthesis process is safer, cost
Also low.
Biological synthesis process is mainly the glutamate decarboxylase (Glutamate decarboxylase, GAD) for utilizing organism
Decarboxylic reaction occurs for the α-carboxyl for being catalyzed Pidolidone or Pidolidone salt, to generate GABA.Its product have it is at low cost, contain
The advantages of amount is high and can be used safely in food, but the usually more difficult acquisition of efficient microorganism fungus kind.Existing document passes through saliva chain
Coccus thermophilous subspecies, Pediococcus pentosaceus, enterococcus faecium, short and small lactobacillus, Bacillus coli cells conversion method prepare the report of GABA.
But since free cell is not easily recycled reuse, there is still a need for the continuous cells for repeating to cultivate high GAD vigor to be used to give birth to
It produces, and by immobilization technology, the cells are fixed or is embedded on solid-state carrier, that is, is prepared into immobilized cell, then can overcome trip
Cellifugal disadvantage, and can realize continuous production.
Zhao Jinglian etc. is reported in digest (bioengineering journal, 1989,5 (2): 124-128) with calcium alginate embedded
Immobilized cell is made in Bacillus coli cells by method, carries out intermittent reaction with 1% glutamic acid solution, the formula of continuously stirring is reacted and is connected
Continuous pillar reaction production GABA.Your equality of chapter digest (Changsha Institute of Electric Power Engineering journal (natural science edition), 1998,13 (4):
It is reported in 433-435) and immobilized cell is made in Bacillus coli cells with calcium alginate embedded method, rear road sodium glutamate mother liquid is mentioned
Waste liquid after taking glutamic acid carries out conversion production GABA.Yang Sheng is far equal public in Chinese patent (patent No. 200910114016.4)
It has opened and has immobilized L. lactis cells using sodium alginate, glutamic acid or paddy ammonia are converted by immobilized cell technology
Sour sodium produces GABA.Jiao Yang, Wang Jianmin and Yang Shengyuan etc. are in digest (nuclear agricultural science report, 2009,23 (6): 1026-1031) with saliva
Liquid chain coccus thermophilous subspecies Y-2 is to have investigated the materials such as carragheen, gelatin and calcium alginate for this bacterial strain immobilization for trying bacterium
Effect, and by comparing the GAD of immobilized cell activity and the yield and carrier mechanical strength of γ-aminobutyric acid, it is determined that sea
Appropriate carrier of the calcium alginate as immobilized cell.CN201010167058.7 provides the fixed plant soft bacillus system of sodium alginate
The method of standby GABA.Sodium alginate is all made of in these methods, but its Resistance to microbes performance is poor, mechanical strength is lower, together
When sodium alginate under the repeated stock of the phosphate buffer of high concentration, can soften, adhesion.
Summary of the invention
In view of the problems of the existing technology, the purpose of the present invention is to provide a kind of preparation methods of fixing lactic acid bacteria
And application, fixing lactic acid bacteria good biocompatibility that this method is prepared, safe and non-toxic, vigor is high, load capacity is big, is catalyzed
It is long to react the service life.It is a further object of the present invention to provide application of the fixing lactic acid bacteria in preparation γ-aminobutyric acid
The technical solution adopted by the present invention to solve the technical problems is:
A kind of preparation method and application of fixing lactic acid bacteria, preparation method the following steps are included:
S1, making lactic acid bacterium concentrate: by the lactic acid bacteria suspension centrifugal concentrating after enrichment culture, obtaining bacterial population is 1
×108~1 × 109The lactic acid bacterium concentrate of CFU/ml；
S2, lactic acid bacteria are fixed:
1) sericin is dissolved with ultrapure water, obtains sericin solution, sericin solution and step S1 is prepared into
The lactic acid bacterium concentrate arrived is uniformly mixed, and obtains liquid A；
2) liquid A obtained to the step 1) instills crosslinking agent, after mixing well at 10-40 DEG C, is crosslinked 1h-20h,
Obtain the sericin hydrogel containing lactic acid bacteria；
3) cellulose powder is dissolved in the cellulose solution that water is swollen, the sericin water-setting obtained to the step 2)
The cellulose solution through being swollen is added in glue, is crosslinked 1~30h after mixing under 20~40 DEG C of stirring conditions, is filtered out with gauze solid
Surely change particle；
4) that crosslinked fluid that step 3) obtains slowly is instilled the calcium chloride that mass percentage concentration is 1-3% by constant flow pump is molten
It in liquid, in 20-40 DEG C of thermotonus 1-4h, then filters, deionized water washing, obtains fixing lactic acid bacteria, 4 DEG C of refrigerators are protected
It deposits.
Preferably, the mass percentage concentration of sericin solution is 1-3% in the step 1).
Preferably, the crosslinking agent in the step 2) is the solution of the glucan containing more aldehyde radicals.
Preferably, the mass percentage concentration of the crosslinking agent in the step 2) is 20-25%.
Preferably, the cellulose in the step 3) is cellulose acetate or carboxymethyl cellulose or DEAE fiber
Element.
Preferably, the concentration of the cellulose in the step 3) is 1-3%.
Preferably, lactic acid bacterium concentrate in the step S2, sericin solution, cellulose solution and containing more aldehyde radicals
Oxidized dextran solution volume ratio be 1~5:1~20:1~20:2~20.
Preferably, lactic acid bacterium concentrate in the step S2, sericin solution, cellulose solution and containing more aldehyde radicals
Oxidized dextran solution volume ratio be 2:4:3:3.
Preferably, temperature is 30 DEG C in the step 2), time 5h；Temperature is 36 DEG C in the step 3), when
Between be 10h.
A kind of application according to the lactic acid bacteria described above being prepared in fermentation preparation γ-aminobutyric acid.
Oxidized dextran containing more aldehyde radicals can be made by laxative remedy: weigh glucan (preferably T-
40) 10.0g, sodium metaperiodate 13.5g are dissolved in respectively in 200ml and 150mlpH4.4 phosphate buffer, by high iodine
Acid sodium solution is added in dextran solution, and 1500r/min is protected from light stirring 4.5h at room temperature, and glycerine 4.5ml, room temperature are added immediately
1500r/min continues to stir 15min, reaction mixture is placed in the bag filter for the molecular weight 3500 that shuts off, and is to be situated between with distilled water
4 DEG C of dialysis 48h of matter, dialyzate freeze-drying, obtain oxidized dextran.
Sericin is the byproduct of mulberry silk industry, and as a kind of good biocompatibility, cell adherence is good, nontoxic, nothing
Pollution, non-stimulated, biodegradable native protein, are widely used in biologic medical field and cosmetic field now,
It is rich in amino, carboxyl isoreactivity group.Lactobacteria-containing sericin aqueous solution of the invention is in crosslinking agent (containing more aldehyde radicals
Glucan) under the action of salt resis.Its hydrogel formed not only has porosity, and degradability also has good
Biocompatibility, can carry various kinds of cell and sertoli cell adheres to and proliferation；Furthermore there is good mechanical performance (silk gum water
Gel has mechanical performance more superior than the alginate hydrogel for being widely used in organizational project).Glucan itself possesses nothing
Poison, good water solubility, cheap feature and a kind of raw material for bio-medical material of classics.Oxygen containing more aldehyde radicals
Changing glucan is a kind of macromolecule glucose polymer that natural glucan obtains after sodium periodate oxidation, is used as being crosslinked
Agent can provide stable crosslinking, and biocompatibility is fabulous, will not cause calcification and the toxic reaction of cell.
Cellulose belongs to a kind of polysaccharide of safety of workers, not the still most important component part of plant, and is also micro-
The representative of the outer macromolecular substances of biological cell.The addition of cellulose helps improve the swelling behavior of material, and material can be made further
Hardening, the mechanical performance of reinforcing material.Cellulose has ultra-fine three-dimensional netted porous structure, can be by lactic acid bacteria, sericin
Uniform adsorption is simultaneously firmly integrated to its surface and inside, in addition, there is cellulose better mechanical performance, excellent biology can drop
Solution property and thermal stability.Multiple aldehyde radicals that oxidized dextran contains respectively with the hydroxyl of high reaction activity in cellulose and silk gum egg
Amino in white carries out hemiacetal and schiff base reaction, sericin grafting is led on cellulose macromolecule, being formed has three-dimensional
The fixing lactic acid bacteria of reticular structure.The secondary cross-linking for realizing lactic acid bacteria is fixed, so that lactic acid bacteria is not easily to fall off, cream obtained
Sour bacteria immobilization bioactivity overall stability is good, high mechanical strength, is conducive to maintain higher lactic acid bacteria concentration.
In addition, sericin is a kind of natural macromolecular albumen, mainly it is made of 18 kinds of amino acid, content is preferably silk ammonia
Acid, is secondly aspartic acid and glutamic acid, these three amino acid contents account for 60% or more of sericin total amino acid content.Oxygen
Changing glucan can also degrade, and catabolite is amino acid and polysaccharide.Therefore, sericin and oxidized dextran in the present invention
Certain fermentation substrate can be also provided for lactic acid bacteria, be conducive to the proliferation of bacterium.
The present invention have it is following the utility model has the advantages that
1) lactic acid bacteria is adsorbed on the gel ball of sericin by the present invention first, then carries out secondary cross-linking with cellulose,
The cellulose of amino-containing sericin and hydroxyl is connected using the aldehyde radical of oxidized dextran as intermediate, being prepared has
The fixing lactic acid bacteria of tridimensional network, short with the transformation period, it is convenient that conversion medium liquid is prepared, and interferes in reaction system
The advantages that substance is few, is not easy microbiological contamination, and product is more single, and cell density is high, and thallus can reuse, and product can be easily separated, can
Reach and improve efficiency, reduces the purpose of cost.
2) lactic acid bacteria is fixed using sericin, cellulose and oxidized dextran containing more aldehyde radicals in the present invention, had
Biocompatibility is high, safe and non-toxic, stability is good, promotes the features such as lactic acid bacteria proliferation.
3) process for fixation of lactic acid bacteria provided by the invention is simple, and condition requires mild, it is easy to accomplish, it is obtained solid
Surely changing lactic acid has many advantages, such as that vigor is high, load capacity is big, the catalysis reaction service life is long.
Specific embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
Embodiment 1
1) fixing lactic acid bacteria is prepared, is included the following steps:
S1, making lactic acid bacterium concentrate: by the lactic acid bacteria suspension centrifugal concentrating after enrichment culture, obtaining bacterial population is 1
×108The lactic acid bacterium concentrate of CFU/ml；
S2, lactic acid bacteria are fixed:
Sericin is dissolved with ultrapure water, obtaining mass percentage concentration is 2% sericin aqueous solution, by sericin water
Solution is uniformly mixed with the lactic acid bacterium concentrate that step S1 is prepared, and obtains liquid A；Crosslinking is added dropwise to obtained liquid A
In agent, after mixing well at 36 DEG C, it is crosslinked 8h, obtains the hydrogel containing lactic acid bacteria；Cellulose powder is dissolved in water and obtains quality
The cellulose solution that percentage concentration 1% is swollen, adds it to hydrogel, is crosslinked 5h under 36 DEG C of stirring conditions after mixing；It will
The crosslinked fluid of acquisition is slowly instilled by constant flow pump in the calcium chloride solution that mass percentage concentration is 3%, in 34 DEG C of thermotonuses
Then 3h is filtered, deionized water washing, obtains fixing lactic acid bacteria, and 4 DEG C of refrigerators save.Wherein, lactic acid bacteria suspension, silk gum egg
The volume ratio ratio of white, cellulose and the oxidized dextran containing more aldehyde radicals is 2:4:3:4, and cellulose is cellulose acetate.
2) γ-aminobutyric acid is prepared
Seed culture is carried out in GYP fermentation medium, the inoculum concentration of fixing lactic acid bacteria is 0.5%, and incubation time is
16 hours, thalline were collected by centrifugation for gained seed culture fluid, thallus suspension seed liquor is made with sterile water, including lactic acid bacteria number is
108~109CFU/ml (plate count).It is packed into 50ml fermentation medium in 250ml triangular flask, is inoculated with 0.2% suspension seed
Liquid adds glucose 1% and L-sodium 1% and is used as carbon source, and addition casein 1% is used as nitrogen source, and stationary culture 24 hours,
γ-aminobutyric acid solution is obtained, GABA content is about 400mg/100ml.
Embodiment 2
1) fixing lactic acid bacteria is prepared, is included the following steps:
S1, making lactic acid bacterium concentrate: by the lactic acid bacteria suspension centrifugal concentrating after enrichment culture, obtaining bacterial population is 1
×108The lactic acid bacterium concentrate of CFU/ml；
S2, lactic acid bacteria are fixed:
Sericin is dissolved with ultrapure water, obtaining mass percentage concentration is 3% sericin aqueous solution, by sericin water
Solution is uniformly mixed with the lactic acid bacterium concentrate that step S1 is prepared, and obtains liquid A；Crosslinking is added dropwise to obtained liquid A
In agent, after mixing well at 34 DEG C, it is crosslinked 5h, obtains the hydrogel containing lactic acid bacteria；Cellulose powder is dissolved in water and obtains quality
The cellulose solution that percentage concentration 2% is swollen, adds it to hydrogel, is crosslinked 10h under 36 DEG C of stirring conditions after mixing；It will
The crosslinked fluid of acquisition is slowly instilled by constant flow pump in the calcium chloride solution that mass percentage concentration is 2%, in 35 DEG C of thermotonuses
Then 2h is filtered, deionized water washing, obtains fixing lactic acid bacteria, and 4 DEG C of refrigerators save.Wherein, lactic acid bacteria suspension, silk gum egg
The volume ratio ratio of white, cellulose and the oxidized dextran containing more aldehyde radicals is 3:3:4:9, and cellulose is carboxymethyl cellulose.
2) γ-aminobutyric acid is prepared
Seed culture is carried out in GYP fermentation medium, the inoculum concentration of fixing lactic acid bacteria is 0.5%, and incubation time is
16 hours, thalline were collected by centrifugation for gained seed culture fluid, and thallus suspension seed liquor is made with sterile water, includes 1 × 109CFU/ml
(plate count).It is packed into 50ml fermentation medium in 250ml triangular flask, is inoculated with 0.2% suspension seed liquor, adds glucose
1% and L-sodium 1% be used as carbon source, addition casein 1% be used as nitrogen source, stationary culture 24 hours, obtain γ-aminobutyric acid
Solution, GABA content are about 350mg/100ml.
3 comparative experiments of embodiment
1) fixing lactic acid bacteria is prepared, is included the following steps:
S1, making lactic acid bacterium concentrate: by the lactic acid bacteria suspension centrifugal concentrating after enrichment culture, obtaining bacterial population is 1
×108The lactic acid bacterium concentrate of CFU/ml；
S2, lactic acid bacteria are fixed:
Sericin is dissolved with ultrapure water, obtaining mass percentage concentration is 1% sericin aqueous solution, by sericin water
Solution is uniformly mixed with the lactic acid bacterium concentrate that step S1 is prepared, and obtains liquid A；Crosslinking is added dropwise to obtained liquid A
In agent, after mixing well at 30 DEG C, it is crosslinked 2h, obtains the hydrogel containing lactic acid bacteria；Cellulose powder is dissolved in water and obtains quality
The cellulose solution that percentage concentration 3% is swollen, adds it to hydrogel, is crosslinked 20h under 36 DEG C of stirring conditions after mixing；It will
The crosslinked fluid of acquisition is slowly instilled by constant flow pump in the calcium chloride solution that mass percentage concentration is 1%, in 35 DEG C of thermotonuses
Then 1h is filtered, deionized water washing, obtains fixing lactic acid bacteria, and 4 DEG C of refrigerators save.Wherein, lactic acid bacteria suspension, silk gum egg
The volume ratio ratio of white, cellulose and the oxidized dextran containing more aldehyde radicals is 1:2:1:3, and cellulose is DEAE cellulose.
2) γ-aminobutyric acid is prepared
Seed culture is carried out in GYP fermentation medium, and it is for 0.5% fixing lactic acid bacteria and not solid to be inoculated with inoculum concentration respectively
Fixed lactic acid bacteria, incubation time are 16 hours, and thalline were collected by centrifugation for gained seed culture fluid, and thallus suspension kind is made with sterile water
Sub- liquid is inoculated in the culture solution of fixing lactic acid bacteria containing 108CFU/ml (plate count), is inoculated in the culture solution of ordinary lactic acid bacteria
Containing 106CFU/ml (plate count).
It is packed into 50ml fermentation medium in 250ml triangular flask, is inoculated with 0.2% above-mentioned suspension seed liquor respectively, adds Portugal
Grape sugar 1% and L-sodium 1% are used as carbon source, and addition casein 1% is used as nitrogen source, stationary culture 24 hours, obtains gamma-amino
Butyric acid solution, being inoculated with GABA content in the culture solution of fixing lactic acid bacteria is about 420mg/100ml, and is inoculated with ordinary lactic acid bacteria
GABA content is about 250mg/100ml in culture solution.
Embodiment 4: the investigation of immobilized cell operational stability
3g immobilized cell is weighed, is suspended in 20mL phosphate buffer (0.2M, pH 6.8), the L- of 1.5g is added
Glu, after 30 DEG C, 160r/min oscillating reactions for 24 hours, amino acid determining instrument measures the alpha-aminobutyric acid content in conversion fluid.It crosses
Immobilized cell is collected in filter, and deionized water is washed twice, is resuspended in 20mL phosphate buffer (0.2M, pH 4.8),
The L-Glu for adding 1.5g, after 30 DEG C, 160r/min oscillating reactions for 24 hours, then in amino acid determining instrument measurement conversion fluid
Alpha-aminobutyric acid content.Conversion ten times is so repeated, immobilized cell stability is preferable, is converted into the first of GABA using L-Glu
Conversion ratio is 100%, is repeated after reacting ten batches, the yield of GABA remains to reach 80% or more, can be by 15.0gL-Glu
It is converted into the GABA of 10.5g, theoretical yield 97.3%.
The preferred embodiments of the invention are only listed above, and protection scope of the present invention is not restricted to this, this field
Made any change is each fallen in the scope of the present invention technical staff within the scope of the invention as claimed.
Claims (10)
1. a kind of preparation method of fixing lactic acid bacteria, which is characterized in that the preparation method comprises the following steps:
S1, making lactic acid bacterium concentrate: by the lactic acid bacteria suspension centrifugal concentrating after enrichment culture, obtaining bacterial population is 1 × 108
~1 × 109The lactic acid bacterium concentrate of CFU/ml；
S2, lactic acid bacteria are fixed:
1) with water-swellable sericin, sericin solution is obtained, the lactic acid that sericin solution and step S1 are prepared
Bacterium concentrate is uniformly mixed, and obtains liquid A；
2) liquid A obtained to the step 1) instills crosslinking agent, is crosslinked 1h- under 10-40 DEG C of stirring condition after mixing well
20h obtains the sericin hydrogel containing lactic acid bacteria；
3) cellulose powder is dissolved in the cellulose solution that water is swollen, into the sericin hydrogel that the step 2) obtains
The cellulose solution through being swollen is added, is crosslinked 1~30h after mixing under 20~40 DEG C of stirring conditions；
4) crosslinked fluid that step 3) obtains slowly is instilled into the calcium chloride solution that mass percentage concentration is 1-3% by constant flow pump
In, it in 20-40 DEG C of thermotonus 1-4h, then filters, deionized water washing, obtains fixing lactic acid bacteria, 4 DEG C of refrigerators save.
2. the preparation method of fixing lactic acid bacteria according to claim 1, which is characterized in that the silk gum in the step 1)
The mass percentage concentration of protein solution is 1-3%.
3. the preparation method of fixing lactic acid bacteria according to claim 1, which is characterized in that the crosslinking in the step 2)
Agent is the solution of the glucan containing more aldehyde radicals.
4. the preparation method of fixing lactic acid bacteria according to claim 3, which is characterized in that the crosslinking in the step 2)
The mass percentage concentration of agent is 20-25%.
5. the preparation method of fixing lactic acid bacteria according to claim 1, which is characterized in that the fiber in the step 3)
Element is cellulose acetate or carboxymethyl cellulose or DEAE cellulose.
6. the preparation method of fixing lactic acid bacteria according to claim 5, which is characterized in that the fiber in the step 3)
The mass percentage concentration of element is 1-3%.
7. the preparation method of fixing lactic acid bacteria according to claim 1, which is characterized in that lactic acid bacteria in the step S2
Concentrate, sericin solution, cellulose solution and the oxidized dextran solution containing more aldehyde radicals volume ratio be 1~5:1~20:
1~20:2~20.
8. the preparation method of fixing lactic acid bacteria according to claim 7, which is characterized in that lactic acid bacteria in the step S2
Concentrate, sericin solution, cellulose solution and the oxidized dextran solution containing more aldehyde radicals volume ratio ratio be 2:4:3:3.
9. the preparation method of fixing lactic acid bacteria according to claim 1, which is characterized in that temperature is in the step 2)
30 DEG C, time 5h；Temperature is 36 DEG C in the step 3), time 10h.
10. the lactic acid bacteria being prepared described in a kind of claim 1-9 any one answering in fermentation preparation γ-aminobutyric acid
With.
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Cited By (2)
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| CN111607539A (en) * | 2020-06-01 | 2020-09-01 | 鲁东大学 | Chinese cabbage extract culture medium and method for producing gamma-aminobutyric acid through fermentation |
| CN115399461A (en) * | 2022-08-30 | 2022-11-29 | 常熟理工学院 | Probiotic sustained-release jelly and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040266992A1 (en) * | 2001-09-11 | 2004-12-30 | Claudio Migliaresi | Method for the preparation of silk fibron hydrogels |
| CN101838672A (en) * | 2010-05-10 | 2010-09-22 | 江南大学 | Method for producing gamma-amino butyric acid by using immobilized lactobacillus plantarum |
-
2018
- 2018-09-08 CN CN201811046703.2A patent/CN109136214B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040266992A1 (en) * | 2001-09-11 | 2004-12-30 | Claudio Migliaresi | Method for the preparation of silk fibron hydrogels |
| CN101838672A (en) * | 2010-05-10 | 2010-09-22 | 江南大学 | Method for producing gamma-amino butyric acid by using immobilized lactobacillus plantarum |
Non-Patent Citations (3)
| Title |
|---|
| CHANG-JIANG LYU ET AL.: "Biosynthesis of g-aminobutyrate by engineered Lactobacillus brevis cells immobilized in gellan gum gel beads", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》 * |
| KI HOON LEE ET AL.: "Sericin-Fixed Silk Fiber as an Immobilization Support of Enzyme", 《FIBERS AND POLYMERS》 * |
| STEFAN IOAN VOICU ET AL.: "Sericin Covalent Immobilization onto Cellulose Acetate Membrane for Biomedical Applications", 《SUSTAINABLE CHEMISTRY AND ENGINERRING》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111607539A (en) * | 2020-06-01 | 2020-09-01 | 鲁东大学 | Chinese cabbage extract culture medium and method for producing gamma-aminobutyric acid through fermentation |
| CN115399461A (en) * | 2022-08-30 | 2022-11-29 | 常熟理工学院 | Probiotic sustained-release jelly and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109136214B (en) | 2020-11-13 |
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