CN109136214B - Preparation method and application of immobilized lactic acid bacteria - Google Patents
Preparation method and application of immobilized lactic acid bacteria Download PDFInfo
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Abstract
The invention discloses a preparation method and application of immobilized lactic acid bacteria, wherein the preparation method comprises the following steps: s1, preparing a lactic acid bacteria concentrated solution: centrifuging and concentrating the lactobacillus suspension after enrichment culture to obtain lactobacillus concentrated solution; s2, lactic acid bacteria fixation: 1) swelling sericin with water to obtain a sericin solution, and uniformly mixing the sericin solution with a lactobacillus concentrated solution to obtain a liquid A; 2) dripping a cross-linking agent into the liquid A obtained in the step 1) to obtain a sericin hydrogel containing lactic acid bacteria; 3) dissolving cellulose powder in water to obtain a swollen cellulose solution, adding the swollen cellulose solution into the sericin hydrogel obtained in the step 2), uniformly mixing and crosslinking; 4) slowly dripping the cross-linking liquid obtained in the step 3) into a calcium chloride solution through a constant flow pump, then filtering, washing with deionized water to obtain immobilized lactobacillus, and storing in a refrigerator at 4 ℃. The immobilized lactobacillus is used for preparing the gamma-aminobutyric acid through fermentation.
Description
Technical Field
The invention belongs to the technical field of immobilized lactic acid bacteria preparation, and particularly relates to a preparation method and application of immobilized lactic acid bacteria.
Background
Gamma-aminobutyric acid (GABA), also known as aminobutyric acid, is a white crystalline powder, a non-proteinogenic amino acid widely distributed in the bodies of animals and plants, and is a major inhibitory neurotransmitter of the central nervous system. The amino group is arranged on the gamma position of butyric acid, exists in an unbound state, is very easy to dissolve in water, is slightly soluble in hot ethanol, is not easy to dissolve in alcohol, ether and phenol, has a sweet taste similar to glutamic acid, and can enhance the flavor of food. The molecular formula is C4H9NO2The molecular weight is 103.12.
GABA can inhibit central nervous system hyperexcitability, has a stabilizing effect on the brain, further promotes relaxation and eliminates nervous tension, and has important physiological functions: (1) improving memory and enhancing memory function; (2) promoting secretion of growth hormone, which has effects of promoting enhancement of skeleton and muscle, enhancing immunity, reducing fat, and improving body function; (3) relieving pain, tranquilizing and improving sleep; (4) has effects of relieving and treating hypertension.
The preparation method of gamma-aminobutyric acid mainly comprises a chemical synthesis method and a biological synthesis method, wherein the chemical synthesis method is mainly characterized in that potassium phthalimide reacts with gamma-chlorobutyronitrile under a strong condition (180 ℃), a product is refluxed and hydrolyzed with concentrated sulfuric acid, and the product is crystallized and purified to obtain the gamma-aminobutyric acid. Although the chemical synthesis of GABA has rapid reaction, the chemical synthesis method has the defects of violent reaction conditions, strong retest, high energy consumption, high cost, more side reactions, serious environmental pollution and the like. Compared with the prior art, the biological synthesis method is safer and has low cost.
The biosynthesis method mainly utilizes Glutamate decarboxylase (GAD) of organisms to catalyze decarboxylation of L-glutamic acid or alpha-carboxyl of L-Glutamate, thereby generating GABA. The product has the advantages of low cost, high content and safe application to food, but high-efficiency microbial strains are generally difficult to obtain. The GABA is prepared by a cell transformation method of streptococcus salivarius subsp thermophilus, pediococcus pentosaceus, enterococcus faecium, lactobacillus brevis and escherichia coli. However, since the free cells are not easy to be recovered and reused, the cells with high GAD activity still need to be cultured repeatedly for production, and the cells are fixed or embedded on a solid carrier by an immobilization technology, i.e. prepared into immobilized cells, so that the defects of the free cells can be overcome, and continuous production can be realized.
Zhaojing et al reported in Abstract (journal of bioengineering, 1989,5 (2): 124-. Octopus et al reported in Abstract (proceedings of the Changsha electric Power college (Nature science edition), 1998,13 (4): 433-. Yangyuan et al, Chinese patent No. 200910114016.4, discloses the production of GABA by immobilizing lactococcus lactis cells with sodium alginate and converting glutamic acid or sodium glutamate by immobilized cell technology. Pyrolunia, Wangjianmin, Yangyuan and the like in an abstract (nuclear agriculture report, 2009, 23(6): 1026) 1031) take streptococcus salivarius subsp thermophilus Y-2 as test bacteria, the effect of immobilizing the strain by materials such as carrageenan, gelatin and calcium alginate is investigated, and the calcium alginate is determined to be a suitable carrier for immobilized cells by comparing the GAD activity of the immobilized cells, the yield of gamma-aminobutyric acid and the mechanical strength of the carrier. CN201010167058.7 provides a method for preparing GABA by immobilizing soft bacillus plantarum with sodium alginate. Sodium alginate is adopted in the methods, but the methods have poor antimicrobial decomposition performance and low mechanical strength, and the sodium alginate can soften and adhere under the repeated impact of high-concentration phosphate buffer.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a preparation method and application of immobilized lactic acid bacteria. Another object of the present invention is to provide the use of immobilized lactic acid bacteria for preparing gamma-aminobutyric acid
The technical scheme adopted by the invention for solving the technical problems is as follows:
a preparation method and application of immobilized lactic acid bacteria are disclosed, wherein the preparation method comprises the following steps:
s1, preparing a lactic acid bacteria concentrated solution: centrifuging and concentrating the suspension of lactobacillus after enrichment culture to obtain lactobacillus with bacteria number of 1 × 108～1×109CFU/ml lactobacillus concentrated solution;
s2, lactic acid bacteria fixation:
1) dissolving sericin with ultrapure water to obtain a sericin solution, and uniformly mixing the sericin solution with the lactic acid bacteria concentrated solution prepared in the step S1 to obtain a liquid A;
2) dripping a cross-linking agent into the liquid A obtained in the step 1), fully and uniformly mixing, and then crosslinking for 1-20 h at 10-40 ℃ to obtain the sericin hydrogel containing the lactic acid bacteria;
3) dissolving cellulose powder in water to obtain a swollen cellulose solution, adding the swollen cellulose solution into the sericin hydrogel obtained in the step 2), uniformly mixing, crosslinking for 1-30 h under the condition of stirring at 20-40 ℃, and filtering out immobilized particles by using gauze;
4) slowly dripping the crosslinking liquid obtained in the step 3) into a calcium chloride solution with the mass percentage concentration of 1-3% through a constant flow pump, reacting for 1-4h at the temperature of 20-40 ℃, then filtering, washing with deionized water to obtain immobilized lactobacillus, and storing in a refrigerator at 4 ℃.
Preferably, the mass percentage concentration of the sericin solution in the step 1) is 1-3%.
Preferably, the cross-linking agent in step 2) is a solution of dextran containing multiple aldehyde groups.
Preferably, the mass percentage concentration of the cross-linking agent in the step 2) is 20-25%.
Preferably, the cellulose in step 3) is cellulose acetate, carboxymethyl cellulose or DEAE cellulose.
Preferably, the concentration of the cellulose in the step 3) is 1-3%.
Preferably, the volume ratio of the lactobacillus concentrated solution, the sericin solution, the cellulose solution and the oxidized dextran solution containing aldehyde groups in the step S2 is 1-5: 1-20: 1-20: 2-20.
Preferably, the volume ratio of the lactic acid bacteria concentrated solution, the sericin solution, the cellulose solution and the oxidized dextran solution containing aldehyde groups in the step S2 is 2: 4: 3: 3.
preferably, the temperature in the step 2) is 30 ℃, and the time is 5 h; the temperature in the step 3) is 36 ℃, and the time is 10 hours.
Application of the lactic acid bacteria prepared according to the method in preparation of gamma-aminobutyric acid through fermentation.
Oxidized dextran containing multiple aldehyde groups can be prepared by the following method: weighing dextran (preferably T-
40)10.0g and 13.5g of sodium periodate are respectively dissolved in 200ml and 150ml of phosphate buffer solution with the pH value of 4.4, the sodium periodate solution is added into the glucan solution, the mixture is stirred for 4.5 hours at the room temperature at 1500r/min in the dark, 4.5ml of glycerol is immediately added, the mixture is continuously stirred for 15 minutes at the room temperature at 1500r/min, the reaction mixed solution is placed in a dialysis bag with the cut-off molecular weight of 3500, the dialysis is carried out for 48 hours at the temperature of 4 ℃ by taking distilled water as a medium, and the dialyzate is frozen and dried to obtain the oxidized glucan.
Sericin is a by-product of silk industry, is a natural protein with good biocompatibility, good cell adhesion, no toxicity, no pollution, no irritation and biodegradability, is widely applied to the fields of biological medicine and cosmetics at present, and is rich in active groups such as amino groups, carboxyl groups and the like. The sericin aqueous solution containing lactic acid bacteria of the invention is crosslinked into gel under the action of a crosslinking agent (dextran containing polyaldehyde groups). The hydrogel formed by the method has porosity, degradability and good biocompatibility, can carry various cells and support cell adhesion and proliferation; in addition, has good mechanical properties (sericin hydrogel has more excellent mechanical properties than alginate hydrogel widely used in tissue engineering). The glucan has the characteristics of no toxicity, good water solubility and low price, and is a classical raw material for biomedical materials. The oxidized dextran containing multiple aldehyde groups is a high molecular glucose polymer obtained by oxidizing natural dextran with sodium periodate, can provide stable crosslinking by using the oxidized dextran as a crosslinking agent, has excellent biocompatibility, and does not cause calcification and toxic reaction of cells.
Cellulose is a worker-safe polysaccharide, not only the major constituent of plants, but also representative of macromolecular substances outside the cells of microorganisms. The addition of the cellulose is helpful for improving the swelling performance of the material, further hardening the material and enhancing the mechanical performance of the material. The cellulose has a superfine three-dimensional reticular porous structure, can uniformly adsorb and firmly combine lactic acid bacteria and sericin on the surface and the inside of the cellulose, and has better mechanical property, excellent biodegradability and thermal stability. A plurality of aldehyde groups contained in the oxidized glucan respectively react with high-reactivity hydroxyl in cellulose and amino in sericin through hemiacetal and Schiff base, and the sericin is grafted on cellulose macromolecules to form the immobilized lactic acid bacteria with a three-dimensional network structure. The secondary cross-linking and fixing of the lactic acid bacteria are realized, the lactic acid bacteria are not easy to fall off, and the prepared immobilized biological activity of the lactic acid bacteria has good overall stability and high mechanical strength, and is beneficial to maintaining higher concentration of the lactic acid bacteria.
In addition, sericin is a natural macromolecular protein, mainly composed of 18 amino acids, preferably serine, and secondly aspartic acid and glutamic acid, the contents of these three amino acids accounting for more than 60% of the total amino acid content of sericin. Oxidized dextrans are also capable of degradation, the degradation products being amino acids and polysaccharides. Therefore, in the invention, the sericin and the oxidized glucan can also provide a certain fermentation substrate for the lactic acid bacteria, and are beneficial to the proliferation of the bacteria.
The invention has the following beneficial effects:
1) the invention firstly adsorbs the lactic acid bacteria to the gel balls of the sericin, then carries out secondary cross-linking with the cellulose, takes the aldehyde group of the oxidized glucan as an intermediate to connect the sericin containing amino and the cellulose containing hydroxyl, and prepares the immobilized lactic acid bacteria with a three-dimensional reticular structure.
2) The invention utilizes sericin, cellulose and oxidized dextran containing multi-aldehyde group to fix the lactobacillus, and has the characteristics of high biocompatibility, safety, no toxicity, good stability, promotion of lactobacillus proliferation and the like.
3) The immobilization method of the lactic acid bacteria provided by the invention is simple, the requirements on conditions are mild, the immobilization method is easy to realize, and the obtained immobilized lactic acid has the advantages of high activity, large loading capacity, long catalytic reaction life and the like.
Detailed Description
The invention is further illustrated by the following specific examples.
Example 1
1) The preparation of the immobilized lactic acid bacteria comprises the following steps:
s1, preparing a lactic acid bacteria concentrated solution: centrifuging and concentrating the suspension of lactobacillus after enrichment culture to obtain lactobacillus with bacteria number of 1 × 108CFU/ml lactobacillus concentrated solution;
s2, lactic acid bacteria fixation:
dissolving sericin with ultrapure water to obtain a sericin aqueous solution with the mass percentage concentration of 2%, and uniformly mixing the sericin aqueous solution with the lactobacillus concentrated solution prepared in the step S1 to obtain a liquid A; dripping the obtained liquid A into a cross-linking agent, fully and uniformly mixing, and then crosslinking for 8 hours at 36 ℃ to obtain the hydrogel containing the lactic acid bacteria; dissolving cellulose powder in water to obtain a swollen cellulose solution with the mass percentage concentration of 1%, adding the swollen cellulose solution into hydrogel, uniformly mixing, and crosslinking for 5 hours at the temperature of 36 ℃ under the stirring condition; slowly dripping the obtained cross-linking solution into a calcium chloride solution with the mass percentage concentration of 3% through a constant flow pump, reacting for 3 hours at the temperature of 34 ℃, then filtering, washing with deionized water to obtain immobilized lactobacillus, and storing in a refrigerator at the temperature of 4 ℃. Wherein the volume ratio of the lactobacillus suspension to the sericin to the cellulose to the oxidized glucan containing multiple aldehyde groups is 2: 4: 3: and 4, the cellulose is cellulose acetate.
2) Preparation of gamma-aminobutyric acid
Performing seed culture in a GYP fermentation culture medium, wherein the inoculation amount of immobilized lactic acid bacteria is 0.5%, the culture time is 16 hours, centrifugally collecting thalli from the obtained seed culture solution, preparing thalli suspension seed solution by using sterile water, and the number of lactic acid bacteria in the seed suspension seed solution is 108-109CFU/ml (plate count). 50ml of fermentation medium is filled into a 250ml triangular flask, 0.2% of suspension seed liquid is inoculated, 1% of glucose and 1% of L-sodium glutamate are added as carbon sources, 1% of casein is added as a nitrogen source, and the mixture is kept stand and cultured for 24 hours to obtain gamma-aminobutyric acid solution, wherein the GABA content is about 400mg/100 ml.
Example 2
1) The preparation of the immobilized lactic acid bacteria comprises the following steps:
s1, preparing a lactic acid bacteria concentrated solution: centrifuging and concentrating the suspension of lactobacillus after enrichment culture to obtain lactobacillus with bacteria number of 1 × 108CFU/ml lactobacillus concentrated solution;
s2, lactic acid bacteria fixation:
dissolving sericin with ultrapure water to obtain a sericin aqueous solution with the mass percentage concentration of 3%, and uniformly mixing the sericin aqueous solution with the lactobacillus concentrated solution prepared in the step S1 to obtain a liquid A; dripping the obtained liquid A into a cross-linking agent, fully and uniformly mixing, and then crosslinking for 5 hours at 34 ℃ to obtain the hydrogel containing the lactic acid bacteria; dissolving cellulose powder in water to obtain a swollen cellulose solution with the mass percentage concentration of 2%, adding the swollen cellulose solution into hydrogel, uniformly mixing, and crosslinking for 10 hours at 36 ℃ under the stirring condition; slowly dripping the obtained cross-linking solution into a calcium chloride solution with the mass percentage concentration of 2% through a constant flow pump, reacting for 2h at the temperature of 35 ℃, then filtering, washing with deionized water to obtain immobilized lactobacillus, and storing in a refrigerator at the temperature of 4 ℃. Wherein the volume ratio of the lactobacillus suspension to the sericin to the cellulose to the oxidized glucan containing multiple aldehyde groups is 3: 4: and 9, the cellulose is carboxymethyl cellulose.
2) Preparation of gamma-aminobutyric acid
Seed culture in GYP fermentation medium, inoculation of immobilized lactic acid bacteriaThe amount of the seed culture was 0.5%, the culture time was 16 hours, the cells were collected by centrifugation, and a cell suspension seed solution containing 1X 10 cells was prepared from the resulting seed culture9CFU/ml (plate count). 50ml of fermentation medium is filled into a 250ml triangular flask, 0.2% of suspension seed liquid is inoculated, 1% of glucose and 1% of L-sodium glutamate are added as carbon sources, 1% of casein is added as a nitrogen source, and the mixture is kept stand and cultured for 24 hours to obtain gamma-aminobutyric acid solution, wherein the GABA content is about 350mg/100 ml.
Example 3 comparative experiment
1) The preparation of the immobilized lactic acid bacteria comprises the following steps:
s1, preparing a lactic acid bacteria concentrated solution: centrifuging and concentrating the suspension of lactobacillus after enrichment culture to obtain lactobacillus with bacteria number of 1 × 108CFU/ml lactobacillus concentrated solution;
s2, lactic acid bacteria fixation:
dissolving sericin with ultrapure water to obtain a sericin aqueous solution with the mass percentage concentration of 1%, and uniformly mixing the sericin aqueous solution with the lactobacillus concentrated solution prepared in the step S1 to obtain a liquid A; dripping the obtained liquid A into a cross-linking agent, fully and uniformly mixing, and then crosslinking for 2h at the temperature of 30 ℃ to obtain the hydrogel containing the lactic acid bacteria; dissolving cellulose powder in water to obtain a swollen cellulose solution with the mass percentage concentration of 3%, adding the swollen cellulose solution into hydrogel, uniformly mixing, and crosslinking for 20 hours at 36 ℃ under the stirring condition; slowly dripping the obtained cross-linking solution into a calcium chloride solution with the mass percentage concentration of 1% through a constant flow pump, reacting for 1h at the temperature of 35 ℃, then filtering, washing with deionized water to obtain immobilized lactobacillus, and storing in a refrigerator at the temperature of 4 ℃. . Wherein the volume ratio of the lactobacillus suspension to the sericin to the cellulose to the oxidized glucan containing multiple aldehyde groups is 1: 2: 1: 3, the cellulose is DEAE cellulose.
2) Preparation of gamma-aminobutyric acid
Performing seed culture in GYP fermentation medium, inoculating immobilized lactobacillus and non-immobilized lactobacillus with inoculum size of 0.5%, respectively, culturing for 16 hr, centrifuging the obtained seed culture solution, collecting thallus, preparing thallus suspension seed solution with sterile water, and inoculating immobilized lactobacillus with culture solution containing 10% of immobilized lactobacillus8CFU/ml (plate count)) The culture solution inoculated with lactobacillus plantarum contains 106CFU/ml (plate count).
50ml of fermentation medium was placed in a 250ml triangular flask, and 0.2% of the above suspension seed solution was inoculated, respectively, 1% of glucose and 1% of L-sodium glutamate were added as carbon sources, 1% of casein was added as nitrogen source, and the mixture was left to stand for 24 hours to obtain a gamma-aminobutyric acid solution, the GABA content in the culture solution inoculated with immobilized lactic acid bacteria was about 420mg/100ml, and the GABA content in the culture solution inoculated with ordinary lactic acid bacteria was about 250mg/100 ml.
Example 4: examination of stability of immobilized cell operation
3g of immobilized cells were weighed, suspended in 20mL of phosphate buffer (0.2M, pH 6.8), added with 1.5g of L-Glu, reacted at 30 ℃ under shaking at 160r/min for 24 hours, and then the content of gamma-aminobutyric acid in the conversion solution was measured with an amino acid analyzer. The immobilized cells were collected by filtration, washed twice with deionized water, resuspended in 20mL of phosphate buffer (0.2M, pH 4.8), added with 1.5g of L-Glu, reacted at 30 ℃ under shaking at 160r/min for 24h, and then the content of gamma-aminobutyric acid in the conversion solution was determined using an amino acid analyzer. The transformation is repeated for ten times, the stability of the immobilized cells is good, the initial transformation rate of converting the L-Glu into the GABA is 100%, the yield of the GABA can still reach more than 80% after repeating ten batches of reactions, 15.0g of the L-Glu can be converted into 10.5g of the GABA, and the theoretical transformation rate is 97.3%.
The above is merely an illustration of the preferred embodiments of the invention, and the scope of the invention is not limited thereto, and any modifications made by those skilled in the art within the scope of the claims of the invention fall within the scope of the invention.
Claims (10)
1. A preparation method of immobilized lactic acid bacteria is characterized by comprising the following steps:
s1, preparing a lactic acid bacteria concentrated solution: centrifuging and concentrating the suspension of lactobacillus after enrichment culture to obtain lactobacillus with bacteria number of 1 × 108～1×109CFU/ml lactobacillus concentrated solution;
s2, lactic acid bacteria fixation:
1) swelling sericin with water to obtain a sericin solution, and uniformly mixing the sericin solution with the lactobacillus concentrated solution prepared in the step S1 to obtain a liquid A;
2) dripping a cross-linking agent into the liquid A obtained in the step 1), fully and uniformly mixing, and then cross-linking for 1-20 h at the temperature of 10-40 ℃ under the stirring condition to obtain the sericin hydrogel containing the lactic acid bacteria;
3) dissolving cellulose powder in water to obtain a swollen cellulose solution, adding the swollen cellulose solution into the sericin hydrogel obtained in the step 2), uniformly mixing, and crosslinking for 1-30 h at 20-40 ℃ under a stirring condition;
4) slowly dripping the crosslinking solution obtained in the step 3) into a calcium chloride solution with the mass percentage concentration of 1-3% through a constant flow pump, reacting for 1-4h at the temperature of 20-40 ℃, then filtering, washing with deionized water to obtain immobilized lactobacillus, and storing in a refrigerator at 4 ℃.
2. The method for preparing an immobilized lactic acid bacterium according to claim 1, wherein the concentration of the sericin solution in the step 1) is 1 to 3% by mass.
3. The method for preparing an immobilized lactic acid bacterium according to claim 1, wherein the cross-linking agent in step 2) is a solution of dextran containing multiple aldehyde groups.
4. The method for producing an immobilized lactic acid bacterium according to claim 3, wherein the concentration of the crosslinking agent in step 2) is 20 to 25% by mass.
5. The method for producing an immobilized lactic acid bacterium according to claim 1, wherein the cellulose in step 3) is cellulose acetate, carboxymethyl cellulose, or DEAE cellulose.
6. The method for producing an immobilized lactic acid bacterium according to claim 5, wherein the cellulose is present in the step 3) at a concentration of 1 to 3% by mass.
7. The method for preparing immobilized lactic acid bacteria according to claim 1, wherein the volume ratio of the lactic acid bacteria concentrate, the sericin solution, the cellulose solution and the oxidized dextran solution containing aldehyde groups in step S2 is 1-5: 1-20: 1-20: 2 to 20.
8. The method for preparing an immobilized lactic acid bacterium according to claim 7, wherein the volume ratio of the lactic acid bacterium concentrate, the sericin solution, the cellulose solution and the oxidized dextran solution containing multiple aldehyde groups in the step S2 is 2: 4: 3: 3.
9. the method for preparing immobilized lactic acid bacteria according to claim 1, wherein the temperature in step 2) is 30 ℃ and the time is 5 hours; the temperature in the step 3) is 36 ℃, and the time is 10 hours.
10. Use of a lactic acid bacterium prepared according to any one of claims 1 to 9 for the fermentative preparation of gamma-aminobutyric acid.
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| Biosynthesis of g-aminobutyrate by engineered Lactobacillus brevis cells immobilized in gellan gum gel beads;Chang-Jiang Lyu et al.;《Journal of Bioscience and Bioengineering》;20190207;第128卷(第2期);123-128 * |
| Sericin Covalent Immobilization onto Cellulose Acetate Membrane for Biomedical Applications;Stefan Ioan Voicu et al.;《Sustainable Chemistry and Enginerring》;20161231;第4卷(第3期);1765-1774 * |
| Sericin-Fixed Silk Fiber as an Immobilization Support of Enzyme;Ki Hoon Lee et al.;《Fibers and Polymers》;20051231;第6卷(第1期);第1页右栏-第2页左栏第1段 * |
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