CN103146673B - Method for preparing microbial nisin - Google Patents
Method for preparing microbial nisin Download PDFInfo
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- CN103146673B CN103146673B CN201310065818.7A CN201310065818A CN103146673B CN 103146673 B CN103146673 B CN 103146673B CN 201310065818 A CN201310065818 A CN 201310065818A CN 103146673 B CN103146673 B CN 103146673B
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- acid bacteria
- lactic acid
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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 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 proteinoid with anti-microbial effect that a class is produced by lactobacillus-fermented or the polypeptides matter passing through processing, and can suppress or kill most Gram-positive pathogenic bacterium, minority is effective to Gram-negative bacteria.Research through forefathers finds, although it can not be used as microbiotic, can be used as food preservatives, and as food preservatives, it has and can not accumulate in vivo and cause untoward reaction, can reduce sterilization temperature, reduces the plurality of advantages such as heat treatment time.Up to the present, unique business-like lactobacillin only has Nisin (a kind of natural bioactive antibacterial peptide of nisin), and Nisin itself has thermostability, and the advantage such as acidproof, low temperature resistant storage.Adopt milk-acid bacteria to ferment during the industrial production of current Nisin under certain condition, to extract and processing obtains.Nisin molecule is in acid, and more soluble in acid substrate, pH rises, and solvability and thermostability all decline.During fermentative production, milk-acid bacteria also can produce lactic acid, lactic acid is a kind of weak acid, along with the accumulation of Lactic Acid from Fermentation Broth and Nisin, the pH of system can decline, and can produce certain injury, make Nisin production declining to lactic-acid bacteria cells, if regulate fermented liquid pH to suitable thalli growth, then the solvability of Nisin and stability can decline.
Immobilized cell technique be able to make up the defect in above-mentioned production.And the current immobilized research for milk-acid bacteria is in the laboratory exploratory stage, adopt entrapping method more, solid support material multiselect natural organic supports material, as alginates, carrageenin, cellulose and its derivates, agar etc., these material common mechanical intensity are lower, mass transfer is poor, affects the supply of nutritive substance and the derivation of product; In addition, Nisin itself to be a kind of molecular weight be 3510 polypeptide, therefore the diffusion in gel likely can be subject to larger impact, cause the Nisin of accumulation higher concentration in gel, thus Nisin is synthesized in lactic-acid bacteria cells growth and continuation in suppression gel, immobilized cell fermentation liquid pH is declined, and Nisin tires reduction, makes the immobilized cell mode of production close to free cell fermentation level; And immobilized cell forming operation is loaded down with trivial details, easy microbiological contamination in process, and serialization industrial production can not be realized.
Summary of the invention
The object of the invention is the present situation that change milk-acid bacteria immobilization production Nisin stagnates on large-scale production road; thering is provided a kind of take absorption method as the whole set process flow process of process for fixation; realize immobilized continuous prodution and operation readiness; and can be used for studying the immobilized continuous prodution effect of milk-acid bacteria, for other microbial immobilized mass-producings and standardization production provide foundation.
The present invention adopts the polyurethane foam of full open aperture as fixation support material simultaneously in conjunction with corresponding reactor; immobilized cell technology; can Cell protection by the impact of extraneous unfavourable condition (as acid, alkali, harmful ion etc.); mass transfer, the supply of nutritive substance and the derivation of product is not affected yet; keep bacterial classification high reactivity simultaneously, and can recycle.Because the Continuous Flow of fermented liquid in Continuous Fermentation Processes adds the timely discharge with tunning Nisin and lactic acid, both the minimizing having reduced fermented liquid in fermenting process is to the restriction of thalli growth and production, and the reduction decreasing the PH that the accumulation due to product causes is on the impact of thalli growth and production.
The present invention is achieved by following technical solution:
Produce a method for microorganism nisin, specifically comprise the following steps:
(1) aperture is that the polyurethane foam carrier material of 30-60PPI is placed in 3%-10% HCl treatment 15-24 hour by solid support material pre-treatment: prepare cation type polyurethane foam carrier;
(2) reactor and material sterilizing: the cation type polyurethane foam carrier of step (1) is filled in reactor, carries out direct-on-line sterilizing; The bottom of this reactor is provided with the sintered glass of microporous medium, as the upholder of fixation support;
(3) inoculate: add lactic acid bacteria culture solution, and press the inoculum size access milk-acid bacteria of 5-10%; Wherein the proportioning of lactic acid bacteria culturing medium is: yeast extract paste 1.5%, peptone 1.5%, potassium primary phosphate 2%, sucrose 1.5%, corn steep liquor 0.3%, halfcystine 0.023%, without iodine refined salt 0.15%, MgSO
47H
2o 0.015%, pH is 7.2;
(4) immobilization: adopt absorption method, by inoculated reactor in 25-35 DEG C of insulation 6-12 hour, milk-acid bacteria raised growth is also adsorbed on polyurethane foam surface;
(5) continuously ferment: collect the fermented liquid in reactor and free thalline; Stream adds fresh medium, coutroi velocity 0.1-2rpm, makes that leavened prod nisin is stable directly to flow out, by the container reception of hydrochloride buffer that PH=2 is housed.
The shape of the solid support material in described step (2) is identical with diameter with the shape of reactor with diameter.
Compared with prior art, tool has the following advantages and effect in the present invention:
1, in the present invention, solid support material is packed in fermenting container after necessarily processing, simple to operate, and sterilizing is convenient.
2, in the present invention, inoculating cell and solid support material add (making) and separately carry out.
3, in the present invention, thalline is fixed and is continuously fermented and to carry out in same reactor.Reduce the probability of living contaminants.
4, can again inoculate after the solid support material used in the present invention is through sterilizing, reusable material.
5, again inoculate in the present invention and do not need carrier again shaping, simple to operate, not easily microbiological contamination.
6, constantly pump into fresh medium in the present invention, release fermented liquid, can substantially keep system environment to be suitable for lactobacter growth, and add a certain amount of acid solution in the container receiving fermented liquid, the Nisin in fermented liquid is stablized, realizes continuous prodution.
Embodiment
Principle of the present invention is that some solid support material has adsorptive power to microorganism cells, can by cell adsorption on its surface, and due to the general porous of these materials, nutrient solution and product mass transfer effect better, are beneficial to the growth of the cell be 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, on cytoactive impact less, and nutrient solution and product mass transfer effect better.
The present invention utilizes has the cation type polyurethane foam of adsorptive power that lactic-acid bacteria cells is absorbed and fixed at its edge surface, pH is made to be that neutral nutrient solution flows in system, for lactic-acid bacteria cells provides fresh nutritive substance, and take away the product Nisin that system pH can be made to decline, damage lactic-acid bacteria cells, realize the continuous prodution 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 calculated by fermenting container volume and working volume etc.
The adition process of above-mentioned fresh medium is realized by peristaltic pump, and control suitable flow rate.
The specific embodiment of the invention is as follows.
Embodiment 1
1. pre-treatment: be that the polyurethane foam carrier material of 30PPI is placed in 3% HCl treatment 24 hours by aperture.
2. sterilizing: be filled in reactor by the solid support material handled well, carries out overall sterilizing.Other line equipment are placed in 121 DEG C in Autoclave, and 20min carries out sterilizing.Then reactor connects reinforced and tapping sebific duct in super clean bench; Outside super clean bench, connect 30 DEG C of circulating water lines and peristaltic pump.
3. reactor inoculation: aseptically, add lactic acid bacteria culture solution in reactor, until do not have solid support material top; The proportioning of lactic acid bacteria culturing medium is: yeast extract paste 1.5%, peptone 1.5%, potassium primary phosphate 2%, sucrose 1.5%, corn steep liquor 0.3%, halfcystine 0.023%, without iodine refined salt 0.15%, MgSO
47H
2o 0.015%, pH is 7.2; Connect bacterium (5% inoculum size).
4. milk-acid bacteria immobilization: have recirculated water to provide 30 DEG C of culture environment after reactor inoculation, then stablize cultivation 8 hours.
5. continuously ferment: after immobilization terminates, open reactor outlet and flow out fermented liquid, drive fresh feed pump, coutroi velocity 1.5rpm, in prolong, squeeze into fresh medium, flow out the interior collection of Erlenmeyer flask of the acid solution of fermented liquid instillation PH=2.Period constantly gets the detection of outflow fermented liquid cell concentration, pH value and Nisin and tires.
Experiment records, and final PH is stabilized in 6.5, and cell concentration also reaches stable, and Nisin tires and reaches 3000IU/ml, and in the continous-stable fermenting process of 13 hours, Nisin tires only to have and slightly declines.
Embodiment 2
1. pre-treatment: be that the polyurethane foam carrier material of 60PPI is placed in 10% HCl treatment 16 hours by aperture.
2. sterilizing: be filled in reactor by the solid support material handled well, carries out overall sterilizing.Other line equipment are placed in 121 DEG C in Autoclave, and 20min carries out sterilizing.Then reactor connects reinforced and tapping sebific duct in super clean bench; Outside super clean bench, connect 35 DEG C of circulating water lines and peristaltic pump.
3. reactor inoculation: aseptically, add lactic acid bacteria culture solution in reactor, until do not have solid support material top; The proportioning of lactic acid bacteria culturing medium is: yeast extract paste 1.5%, peptone 1.5%, potassium primary phosphate 2%, sucrose 1.5%, corn steep liquor 0.3%, halfcystine 0.023%, without iodine refined salt 0.15%, MgSO
47H
2o 0.015%, pH is 7.2; Connect bacterium (10% inoculum size).
4. milk-acid bacteria immobilization: have recirculated water to provide 35 DEG C of culture environment after reactor inoculation, then stablize cultivation 7 hours.
5. continuously ferment: after immobilization terminates, open reactor outlet and flow out fermented liquid, drive fresh feed pump, coutroi velocity 1.5rpm, in prolong, squeeze into fresh medium, flow out the interior collection of Erlenmeyer flask of the acid solution of fermented liquid instillation PH=2.Period constantly gets the detection of outflow fermented liquid cell concentration, pH value and Nisin and tires.
Experiment records, and final PH is stabilized in 7.0, and cell concentration also reaches stable, and Nisin tires and reaches 3500IU/ml, and in the continous-stable fermenting process of 12 hours, Nisin tires and to fluctuate between 3100-3500IU/ml.
Claims (2)
1. produce a method for microorganism nisin, specifically comprise the following steps:
(1) aperture is that the polyurethane foam carrier material of 30-60PPI is placed in 3%-10% HCl treatment 15-24 hour by solid support material pre-treatment: prepare cation type polyurethane foam carrier;
(2) reactor and material sterilizing: the cation type polyurethane foam carrier of step (1) is filled in reactor, carries out direct-on-line sterilizing; The bottom of this reactor is provided with the sintered glass of microporous medium, as the upholder of fixation support;
(3) inoculate: add lactic acid bacteria culture solution, and press the inoculum size access milk-acid bacteria of 5-10%; Wherein the proportioning of lactic acid bacteria culturing medium is: yeast extract paste 1.5%, peptone 1.5%, potassium primary phosphate 2%, sucrose 1.5%, corn steep liquor 0.3%, halfcystine 0.023%, without iodine refined salt 0.15%, MgSO
47H
2o 0.015%, pH is 7.2;
(4) immobilization: adopt absorption method, by inoculated reactor in 25-35 DEG C of insulation 6-12 hour, milk-acid bacteria raised growth is also adsorbed on polyurethane foam surface;
(5) continuously ferment: collect the fermented liquid in reactor and free thalline; Stream adds fresh medium, coutroi velocity 0.1-2rpm, makes that leavened prod nisin is stable directly to flow out, by the container reception of hydrochloride buffer that PH=2 is housed.
2. a kind of method of producing microorganism nisin according to claim 1, is characterized in that: the shape of the solid support material in described step (2) is identical with diameter with the shape of reactor with diameter.
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| 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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| CN103146673B true CN103146673B (en) | 2015-01-21 |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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 |
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 |
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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 |
|---|
| Continuous production of lacticin 3147 and nisin using cell simmobilized in calcium alginate;A.G.M. Scannell等;《Journal of Applied Microbiology》;20001031;第89卷(第4期);574,575,577 * |
| 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;欧阳军梅等;《生物加工过程》;20081130;第6卷(第6期);52 * |
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