CN103146673A - Microbial immobilization method - Google Patents
Microbial immobilization method Download PDFInfo
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- CN103146673A CN103146673A CN2013100658187A CN201310065818A CN103146673A CN 103146673 A CN103146673 A CN 103146673A CN 2013100658187 A CN2013100658187 A CN 2013100658187A CN 201310065818 A CN201310065818 A CN 201310065818A CN 103146673 A CN103146673 A CN 103146673A
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- reactor
- acid bacteria
- nisin
- polyurethane foam
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a microbial immobilization method, comprising immobilization for lactic acid bacteria and a continuous production process for Nisin. The microbial immobilization method comprises the following steps of: firstly, growing thalli in a reactor and adsorbing the thalli onto carrier materials; secondly, releasing the free thalli and adding fresh culture solution, and controlling a flow speed to enable the amount of the product Nisin to be the highest when fermentation solution just arrives at the bottom of the reactor and flows out by combining with the height-diameter ratio of the reactor; and finally, enabling the product to flow in a product collection tank, wherein the carriers used are reusable polyurethane foam carrier materials; the whole process is simple to operate; and because two restrictive factors influencing the growth and the production of lactic acid bacteria, namely, nutritional ingredient reduction and low pH caused by the product, are eliminated, continuous production for lactic acid bacteria under a high Nisin yield can be realized.
Description
Technical field
The invention relates to the microorganism cells immobilization technology, particularly the processing method that can be continuously produced of a kind of new fixing lactic acid bacteria.
Background technology
Lactobacillin is the polypeptides matter that a class is processed by the proteinoid with anti-microbial effect or the process of lactobacillus-fermented generation, can suppress or kill most Gram-positive pathogenic bacteriums, and minority is effective to Gram-negative bacteria.Find through forefathers' research, although it can not be used as microbiotic, can be used as food preservatives, and it has and can not accumulate in vivo and cause untoward reaction as food preservatives, can reduce sterilization temperature, the plurality of advantages such as minimizing heat treatment time.Up to the present, unique business-like lactobacillin only has the natural bioactive antibacterial peptide of a Nisin(nisin), itself has thermostability Nisin, and the advantage such as acidproof, low temperature resistant storage.Adopt at present that milk-acid bacteria is fermented during the industrial production of Nisin under certain condition, extraction and processing obtains.It is acid that the Nisin molecule is, more soluble in acid substrate, and pH rises that solvability and thermostability all descend.During fermentative production, milk-acid bacteria also can produce lactic acid, lactic acid is a kind of weak acid, accumulation along with Lactic Acid from Fermentation Broth and Nisin, the pH of system can descend, and can produce certain injury to lactic-acid bacteria cells, makes the Nisin production declining, if regulate fermented liquid pH to suitable thalli growth, the solvability of Nisin and stability can descend.
Immobilized cell technique be able to make up the defective in above-mentioned production.And current immobilized research for milk-acid bacteria is in the laboratory exploratory stage, the entrapping methods that adopt more, the natural organic support material of solid support material multiselect, as alginates, carrageenin, Mierocrystalline cellulose and derivative, agar etc., these material common mechanical intensity are lower, mass transfer is relatively poor, affects the supply of nutritive substance and the derivation of product; In addition, Nisin itself is that a kind of molecular weight is 3510 polypeptide, therefore the diffusion in gel might be subject to larger impact, cause the Nisin of accumulation higher concentration in gel, thereby the synthetic Nisin of lactic-acid bacteria cells growth and continuation in the inhibition gel, immobilized cell fermentation liquid pH is descended, and the Nisin reduction of tiring makes the immobilized cell mode of production close to the free cell fermentation level; And the immobilized cell forming operation is loaded down with trivial details, easily microbiological contamination in process, and can not realize the serialization industrial production.
Summary of the invention
The objective of the invention is to change the milk-acid bacteria immobilization and produce the present situation that Nisin stagnates on the large-scale production road; a kind of a whole set of technical process take absorption method as process for fixation is provided; realize immobilized serialization production and operation readiness; and can be used for studying the immobilized serialization production effect of milk-acid bacteria, producing with standardization for other microbial immobilized mass-producings provides foundation.
The present invention adopts the polyurethane foam of full open aperture as the corresponding reactor of fixation support material combination simultaneously; immobilized cell technology; can Cell protection be subjected to the impact of extraneous unfavourable condition (as acid, alkali, harmful ion etc.); do not affect the supply of mass transfer, nutritive substance and the derivation of product yet; keep simultaneously the bacterial classification high reactivity, and can recycle.Because the Continuous Flow of fermented liquid in Continuous Fermentation Processes adds timely discharge with tunning Nisin and lactic acid, both lower the minimizing of fermented liquid in the fermenting process to the restriction of thalli growth and production, also reduced the reduction of the pH that the accumulation due to product causes to the impact of thalli growth and production.
The present invention is achieved by following technical solution:
A kind of microorganism immobilization method specifically comprises the following steps:
(1) solid support material pre-treatment: preparation cation type polyurethane foam carrier, the polyurethane foam carrier material that is 30-60PPI with the aperture is placed in 3%-10% salt acid treatment 15-24 hour;
(2) reactor and material sterilization: the cation type polyurethane foam carrier of step (1) is filled in reactor, carries out the direct-on-line sterilization; The bottom of this reactor is provided with microporous medium;
(3) inoculation: add lactic acid bacteria culture solution, and press the inoculum size access milk-acid bacteria of 5-10%; Wherein the proportioning of milk-acid bacteria substratum is: yeast extract paste 1.5%, and peptone 1.5%, potassium primary phosphate 2%, sucrose 1.5%, corn steep liquor 0.3%, halfcystine 0.023% is without iodine refined salt 0.15%, MgSO
47H
2O0.015%, pH are 7.2;
(4) immobilization: adopt absorption method, the reactor inoculate is incubated 6-12 hour in 25-35 ℃, the milk-acid bacteria raised growth also is adsorbed on the polyurethane foam surface;
(5) continuously ferment: collect fermented liquid and free thalline in reactor; Stream adds fresh medium, and coutroi velocity 0.1-2rpm makes the stable directly outflow of leavened prod, by the container reception of the hydrochloride buffer that pH=2 is housed.
The shape of the solid support material in described step (2) is identical with shape and the diameter of reactor with diameter.
Microporous medium in described step (2) is sintered glass, as the upholder of fixation support.
The present invention compared with prior art has following advantage and effect:
1, in the present invention, solid support material is packed in fermenting container after necessarily processing, and is simple to operate, and sterilization is convenient.
2, in the present invention, inoculating cell and solid support material add (making) separately to carry out.
3, in the present invention, thalline is fixed and is continuously fermented and carries out in same reactor.Reduce the probability of living contaminants.
4, the solid support material that uses in the present invention be through the sterilization after can again inoculate, reusable material.
5, in the present invention again the inoculation do not need carrier moulding again, simple to operate, be difficult for microbiological contamination.
6, constantly pump into fresh medium in the present invention, emit fermented liquid, can substantially keep the interior environment of system to be suitable for lactobacter growth, and add a certain amount of acid solution in the container of reception fermented liquid, make the Nisin in fermented liquid stable, realize serialization production.
Embodiment
Principle of the present invention is that some solid support material has adsorptive power to microorganism cells, cell can be adsorbed on its surface, and due to the general porous of these materials, nutrient solution and product mass transfer effect are better, are beneficial to the growth of the cell that is fixed.
The present invention adopts absorption method, utilizes the electrostatic attraction effect between organic porous material and cell surface that cell is fixed on material surface, and is less on cytoactive impact, and nutrient solution and the product mass transfer effect better.
Utilization of the present invention has the cation type polyurethane foam of adsorptive power that lactic-acid bacteria cells is absorbed and fixed at its edge surface, making pH is that neutral nutrient solution flows in system, for lactic-acid bacteria cells provides fresh nutritive substance, and take away can make system pH descend, to the product Nisin that lactic-acid bacteria cells damages, realize the serialization production of Nisin.
Production technique of the present invention comprises: fill solid support material; Add fermentation culture, inoculation, thalline immobilization; Chuck leads to thermostatical circulating water; Pass into fresh medium, receive fermented liquid; Timing sampling detects the parameters such as fermented liquid pH, free cell amount and Nisin tire.
The shape of above-mentioned filling solid support material and volume are to calculate by fermenting container volume and working volume etc.
The adition process of above-mentioned fresh medium is to realize by peristaltic pump, and controls suitable flow velocity.
The specific embodiment of the invention is as follows.
Embodiment 1
1. pre-treatment: the polyurethane foam carrier material that is 30PPI with the aperture is placed in 3% salt acid treatment 24 hours.
2. sterilization: the solid support material of handling well is filled in reactor, carries out the integral body sterilization.It is interior in 121 ℃ that other line equipment are placed in Autoclave, and 20min sterilizes.Then reactor connects reinforced and tapping sebific duct in super clean bench; Outside super clean bench, connect 30 ℃ of circulating water lines and peristaltic pump.
3. reactor inoculation: under aseptic condition, add lactic acid bacteria culture solution in reactor, until do not have the solid support material top; The proportioning of milk-acid bacteria substratum is: yeast extract paste 1.5%, and peptone 1.5%, potassium primary phosphate 2%, sucrose 1.5%, corn steep liquor 0.3%, halfcystine 0.023% is without iodine refined salt 0.15%, MgSO
47H
2O0.015%, pH are 7.2; Connect bacterium (5% inoculum size).
4. milk-acid bacteria immobilization: have recirculated water that 30 ℃ of culture environment are provided after the reactor inoculation, then stable the cultivation 8 hours.
5. continuously ferment: after immobilization finishes, open reactor outlet and flow out fermented liquid, drive fresh feed pump, coutroi velocity 1.5rpm squeezes into fresh medium in prolong, flows out interior collection of Erlenmeyer flask that fermented liquid splashes into the acid solution of pH=2.Constantly getting during this time outflow fermented liquid detection cell concentration, pH value and Nisin tires.
Experiment records, and final pH is stabilized in 6.5, and it is stable that cell concentration also reaches, and Nisin tires and reach 3000IU/ml, and in the continous-stable fermenting process of 13 hours, Nisin tires only to have slightly and descends.
Embodiment 2
1. pre-treatment: the polyurethane foam carrier material that is 60PPI with the aperture is placed in 10% salt acid treatment 16 hours.
2. sterilization: the solid support material of handling well is filled in reactor, carries out the integral body sterilization.It is interior in 121 ℃ that other line equipment are placed in Autoclave, and 20min sterilizes.Then reactor connects reinforced and tapping sebific duct in super clean bench; Outside super clean bench, connect 35 ℃ of circulating water lines and peristaltic pump.
3. reactor inoculation: under aseptic condition, add lactic acid bacteria culture solution in reactor, until do not have the solid support material top; The proportioning of milk-acid bacteria substratum is: yeast extract paste 1.5%, and peptone 1.5%, potassium primary phosphate 2%, sucrose 1.5%, corn steep liquor 0.3%, halfcystine 0.023% is without iodine refined salt 0.15%, MgSO
47H
2O0.015%, pH are 7.2; Connect bacterium (10% inoculum size).
4. milk-acid bacteria immobilization: have recirculated water that 35 ℃ of culture environment are provided after the reactor inoculation, then stable the cultivation 7 hours.
5. continuously ferment: after immobilization finishes, open reactor outlet and flow out fermented liquid, drive fresh feed pump, coutroi velocity 1.5rpm squeezes into fresh medium in prolong, flows out interior collection of Erlenmeyer flask that fermented liquid splashes into the acid solution of pH=2.Constantly getting during this time outflow fermented liquid detection cell concentration, pH value and Nisin tires.
Experiment records, and final pH is stabilized in 7.0, and it is stable that cell concentration also reaches, and Nisin tires and reach 3500IU/ml, and in the continous-stable fermenting process of 12 hours, Nisin tires and fluctuates between 3100-3500IU/ml.
Claims (3)
1. microorganism immobilization method specifically comprises the following steps:
(1) solid support material pre-treatment: preparation cation type polyurethane foam carrier, the polyurethane foam carrier material that is 30-60PPI with the aperture is placed in 3%-10% salt acid treatment 15-24 hour;
(2) reactor and material sterilization: the cation type polyurethane foam carrier of step (1) is filled in reactor, carries out the direct-on-line sterilization; The bottom of this reactor is provided with microporous medium;
(3) inoculation: add lactic acid bacteria culture solution, and press the inoculum size access milk-acid bacteria of 5-10%; Wherein the proportioning of milk-acid bacteria substratum is: yeast extract paste 1.5%, and peptone 1.5%, potassium primary phosphate 2%, sucrose 1.5%, corn steep liquor 0.3%, halfcystine 0.023% is without iodine refined salt 0.15%, MgSO
47H
2O0.015%, pH are 7.2;
(4) immobilization: adopt absorption method, the reactor inoculate is incubated 6-12 hour in 25-35 ℃, the milk-acid bacteria raised growth also is adsorbed on the polyurethane foam surface;
(5) continuously ferment: collect fermented liquid and free thalline in reactor; Stream adds fresh medium, and coutroi velocity 0.1-2rpm makes the stable directly outflow of leavened prod, by the container reception of the hydrochloride buffer that pH=2 is housed.
2. a kind of microorganism immobilization method according to claim 1, it is characterized in that: the shape of the solid support material in described step (2) is identical with shape and the diameter of reactor with diameter.
3. a kind of microorganism immobilization method according to claim 1, it is characterized in that: the microporous medium in described step (2) is sintered glass, as the upholder of fixation support.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310065818.7A CN103146673B (en) | 2013-03-01 | 2013-03-01 | Method for preparing microbial nisin |
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| CN201310065818.7A CN103146673B (en) | 2013-03-01 | 2013-03-01 | Method for preparing microbial nisin |
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| CN103146673A true CN103146673A (en) | 2013-06-12 |
| CN103146673B CN103146673B (en) | 2015-01-21 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103355728A (en) * | 2013-07-01 | 2013-10-23 | 福建省农业科学院农业工程技术研究所 | Immobilized semi-continuous lactic acid fermentation method of juice |
| CN105461879A (en) * | 2015-11-11 | 2016-04-06 | 浙江省林业科学研究院 | Multipurpose polyurethane effective microorganism solidification carrier |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108131A (en) * | 2010-12-29 | 2011-06-29 | 清华大学 | Hydrophilic cationic polyurethane foam plastics and preparation method thereof |
| CN102115546A (en) * | 2010-12-29 | 2011-07-06 | 清华大学 | Preparation method of hydrophilic cation type polyurethane foam plastic by means of direct foaming |
-
2013
- 2013-03-01 CN CN201310065818.7A patent/CN103146673B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108131A (en) * | 2010-12-29 | 2011-06-29 | 清华大学 | Hydrophilic cationic polyurethane foam plastics and preparation method thereof |
| CN102115546A (en) * | 2010-12-29 | 2011-07-06 | 清华大学 | Preparation method of hydrophilic cation type polyurethane foam plastic by means of direct foaming |
Non-Patent Citations (2)
| Title |
|---|
| A.G.M. SCANNELL等: "Continuous production of lacticin 3147 and nisin using cell simmobilized in calcium alginate", 《JOURNAL OF APPLIED MICROBIOLOGY》, vol. 89, no. 4, 31 October 2000 (2000-10-31), pages 573 - 579 * |
| 欧阳军梅等: "聚氨酯泡沫固定产脂肪酶粗状假丝酵母(Candida valida 12)细胞的研究", 《生物加工过程》, vol. 6, no. 6, 30 November 2008 (2008-11-30), pages 52 - 57 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103355728A (en) * | 2013-07-01 | 2013-10-23 | 福建省农业科学院农业工程技术研究所 | Immobilized semi-continuous lactic acid fermentation method of juice |
| CN105461879A (en) * | 2015-11-11 | 2016-04-06 | 浙江省林业科学研究院 | Multipurpose polyurethane effective microorganism solidification carrier |
| CN105461879B (en) * | 2015-11-11 | 2018-01-19 | 浙江省林业科学研究院 | A kind of multipurpose urethane effective microbe solidified carrier |
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| CN103146673B (en) | 2015-01-21 |
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