CN102653733A - Method for producing low-temperature superoxide dismutase through microbial fermentation - Google Patents
Method for producing low-temperature superoxide dismutase through microbial fermentation Download PDFInfo
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- CN102653733A CN102653733A CN2012101067438A CN201210106743A CN102653733A CN 102653733 A CN102653733 A CN 102653733A CN 2012101067438 A CN2012101067438 A CN 2012101067438A CN 201210106743 A CN201210106743 A CN 201210106743A CN 102653733 A CN102653733 A CN 102653733A
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Abstract
The invention discloses a method for producing low-temperature superoxide dismutase through microbial fermentation. The method comprises the following steps of: performing gradual low-temperature acclimation of the microorganism for producing superoxide dismutase so that the microorganism grows well in low-temperature environment; performing gradual amplification culture of the superoxide dismutase producing bacteria after the low-temperature acclimation at 10-16 DEGE C, inoculating the bacteria into a liquid fermentation medium according to an inoculation amount of 3-9% of the fermentation broth volume, and culturing at 10-16 DEG C for 72-144 hours to end the microbial fermentation for producing low-temperature superoxide dismutase; centrifuging the fermentation broth at 4,000-8,000rpm and collecting the bacteria; washing the bacteria for multiple times and collecting the precipitate; suspending the precipitate in a buffer solution and adding quartz sand and grinding; centrifuging at 10,000-14,000rpm and collecting the supernate which is the crude enzyme liquid; and performing further concentration, separation and purification of the crude enzyme liquid according to different needs and different objects to obtain the enzymic preparations different in activity, purity and form.
Description
Technical field
The present invention relates to fields such as microbiology, enzyme engineering, fermentation engineering, biological chemistry, be specifically related to the method that a kind of microbial fermentation is produced SOD.The low temperature SOD that this method is produced is as the sanitising agent of ultra-oxygen anion free radical in the organism; Be mainly used in industries such as medical science, food, makeup, especially demonstrate unique advantages at aspects such as radioprotective, anti-ageing, anti-inflammatory, inhibition tumour, cancer and autoimmunization treatments.
Background technology
Superoxide-dismutase (superoxide dismutase; ECl.15.1.1 is called for short SOD) be one type and extensively be present in biological intravital metalloenzyme, can catalysis ultra-oxygen anion free radical and hydrogen ion reaction formation hydrogen peroxide and molecular oxygen; Thereby remove biological intravital oxyradical; Play the protection organism effect of escaping injury (from pretty blue or green, Dali teachers training school journal, 1998).Mccord in 1969 and Fridovich find the active and called after superoxide-dismutase (Lin Qingbin, chemistry world, 2006) of this enzymes biocatalysis.Different by its bonded metal species, can it be divided three classes: one type is glaucous Cu, Zn-SOD, relative molecular weight is about 32000, mainly is present in eukaryotic cytosol and the chloroplast(id), be study at most at present, the most deep one type; Second type is mauve Mn, Fe-SOD, and relative molecular weight is about 40000, mainly is present in prokaryote and the mitochondrial matrix; The 3rd type is filemot Fe-SOD, and relative molecular weight is about 38700, mainly is present in (Chen Huifang, the chemistry of life, 2003) in prokaryotic cell prokaryocyte and some plants.
SOD is as biological enzyme formulation, is mainly used in auxiliary radiotherapy and chemotherapy in the medical treatment, and radiation-induced spinoff is eliminated in the protection and the transplanting of organs such as kidney, liver, heart, and as the probe of some disease etc.; Foodstuffs industry is applied to beverage and beer etc. as additive.SOD is widely used in treating multiple diseases such as oxygen intoxication, senile cataract, mellitus, cardiovascular disorder, various inflammation (Zhang Borun etc., microbiology circular, 1992) in recent years.SOD studies focus both at home and abroad in decades; Initialization phase focuses mostly in from animal blood or tissue, extracting SOD; The research of producing SOD to the mid-term stage microbial fermentation begins to start to walk and day by day increase, and up to the present microorganisms producing SOD becomes the main body of research already.But be mostly medium and high temperature SOD, optimum temperature is generally more than 45 ℃, and the physiological temp of human body is generally 36.5～37 ℃; Cause the medium and high temperature SOD of microbial fermentation production not play one's part to the full; The result of treatment difference occurs or do not have effect (Ceng Yinxin, JOURNAL OF MICROBIOLOGY, 2004).Low temperature SOD optimum temperature is generally 35～40 ℃, and relatively near the physiological temp of human body, it is apparent in view to be applied to result of treatment.But low temperature SOD industrialization, large-scale production and application also do not appear in the newspapers (Wujiang etc., Chinese Journal of Pharmaceuticals, 1997).Seeing that the limitation of the medium and high temperature SOD optimum temperature that animal blood source difficulty and microbial fermentation are produced utilizes the low temperature SOD of microbial fermentation production to have very big application advantage.Low temperature SOD has high enzymatic activity and high catalytic efficiency (under the physical environment temperature; And to thermo-responsive, the vigor of cold-adapted enzyme is lost through gentle thermal treatment, if thereby SOD is applied to foodstuffs industry to be shortened the time of treating processes greatly and saves expensive heating or refrigeration costs; And do not influence product quality (Zheng Zhou etc.; Polar research, 2007), this will help popularization and the use of low temperature SOD.Can fundamentally break away from heating, cooling apparatus and the flow process of loaded down with trivial details extraction process and medium and high temperature enzyme, when improving yield and enzymic activity, cut down the consumption of energy and production cost.In view of the advantage of low temperature SOD under nature and physiological condition, low temperature SOD has application prospects and potentiality to be exploited more at aspects such as health care, food, makeup.
Summary of the invention
The present invention provides the method that a kind of microbial fermentation is produced low temperature SOD; This method mainly is that mikrobe is through after the domestication by low temperature; Produce the method for low temperature SOD at 10～16 ℃ of liquid fermentings; The low temperature SOD enzymic activity that this working method obtains can reach 203.68U/ml, through separating and purifying, can obtain the zymin of different concns and purity as again.This zymin application operating is simple, convenient, fast, cost is low.Can fundamentally avoid heating, cooling apparatus and the technology of middle temperature, high temperature enzyme.
The method that a kind of microbial fermentation of the present invention is produced low temperature SOD specifically may further comprise the steps:
The mikrobe that (1) will produce SOD is domestication by low temperature step by step, and acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃ → 16 ℃ → 14 ℃ → 12 ℃ → 10 ℃, makes its well-grown in low temperature environment from high to low;
(2) produce 10～16 ℃ of bacterium enlarged culturing step by step by the SOD of ordinary method after, be prepared into liquid first order seed and secondary seed domestication by low temperature;
(3) with liquid first order seed or secondary seed, 3～9% inoculum sizes of pressing fermentating liquid volume insert in the liquid fermentation medium, and when cultivating 68～96h for 10～16 ℃, promptly microbial fermentation is produced low temperature SOD end;
(4) with the fermented liquid 4,000～8 of (3), the centrifugal collection thalline of 000rpm is with centrifugal collecting precipitation after the distilled water wash several;
(5) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collection is the SOD crude enzyme liquid;
(6) different with the use object according to different needs, the crude enzyme liquid that (5) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
The bacterial classification that uses among the present invention derives from China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and initial stage activation and growth conditions are undertaken by the explanation that culture presevation unit provides.SOD produces bacterium (CGMCC numbering: 1.119 or 1.551) after first activation, the domestication by low temperature; By condition of enzyme production fermentative prodn low temperature SOD of the present invention; Bacterial strain after the domestication by low temperature can be preserved 2 months at 4 ℃, bacteria suspension, the preservation for a long time under-80 ℃ of conditions of processing with 10～25% glycerine.
Embodiment
Embodiment one:
(1) medium preparation
1. strain activation and culture base: glucose 10.0g, Carnis Bovis seu Bubali cream 14.0g, peptone 7.0g, yeast powder 4.0g, agar 20.0g, pH 7.0, zero(ppm) water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
2. bacterial classification domestication by low temperature substratum: glucose 8.0～16.0g, Carnis Bovis seu Bubali cream 10.0～25.0g, peptone 3.0～12.0g, yeast powder 2.0～9.0g, agar 10.0～30.0g, zero(ppm) water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
3. liquid seed culture medium: glucose 5.0～15.0g, Carnis Bovis seu Bubali cream 8.0～20.0g, peptone 5.0～15.0g, yeast powder 2.0～10.0g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
4. enzymatic production substratum: glucose 10.0～30.0g, (NH
4)
2SO
43.0～10.0g, steeping water 5.0～15.0g, yeast powder 2.0～8.0g, CaCO
31.0～6.0g, MgSO
40.1～0.9g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
(2) will produce the bacterial classification of SOD, carry out initial activation by the bacterial strain explanation that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) bacterial classification that (2) activation is good domestication by low temperature step by step, acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃ → 16 ℃ → 14 ℃ → 12 ℃ → 10 ℃, makes its well-grown in low temperature environment from high to low;
(4) produce bacterium by the SOD of ordinary method after and cultivate 24～36h at 10～16 ℃ and then carry out enlarged culturing step by step, be prepared into liquid first order seed and secondary seed by 5～8% inoculum size with domestication by low temperature;
(5) inoculum size that the secondary seed that (4) is prepared is pressed fermentating liquid volume 3～5% inserts in the product enzyme substratum of 10L, and when cultivating 120～144h for 10～12 ℃, promptly microbial fermentation is produced low temperature SOD end;
(6) with the fermented liquid 4,000～8 of (5), the centrifugal collection thalline of 000rpm is with centrifugal collecting precipitation after the distilled water wash several;
(7) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the SOD crude enzyme liquid;
(8) different with the use object according to different needs, the crude enzyme liquid that (7) are obtained further concentrates, separation and purification, is prepared into the low temperature SOD preparation of different activities, purity and formulation.
Embodiment two:
(1) medium preparation
1. strain activation and culture base: glucose 10.0g, Carnis Bovis seu Bubali cream 14.0g, peptone 7.0g, yeast powder 4.0g, agar 20.0g, pH 7.0, zero(ppm) water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
2. bacterial classification domestication by low temperature substratum: glucose 8.0～16.0g, Carnis Bovis seu Bubali cream 10.0～25.0g, peptone 3.0～12.0g, yeast powder 2.0～9.0g, agar 10.0～30.0g, zero(ppm) water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
3. liquid seed culture medium: glucose 5.0～15.0g, Carnis Bovis seu Bubali cream 8.0～20.0g, peptone 5.0～15.0g, yeast powder 2.0～10.0g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
4. enzymatic production substratum: glucose 10.0～30.0g, (NH
4)
2SO
43.0～10.0g, steeping water 5.0～15.0g, yeast powder 2.0～8.0g, CaCO
31.0～6.0g, MgSO
40.1～0.9g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
(2) will produce the bacterial classification of SOD, carry out initial activation by the bacterial strain explanation that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) bacterial classification that (2) activation is good domestication by low temperature step by step, acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃ → 16 ℃ → 14 ℃ → 12 ℃ → 10 ℃, makes its well-grown in low temperature environment from high to low;
(4) produce bacterium by the SOD of ordinary method after and cultivate 24～36h, then carry out enlarged culturing step by step, be prepared into liquid first order seed and secondary seed by 5～8% inoculum size at 10～16 ℃ with domestication by low temperature;
(5) inoculum size that the secondary seed that (4) is prepared is pressed fermentating liquid volume 5～7% inserts in the product enzyme substratum of 50L, and when cultivating 96～120h for 12～14 ℃, promptly microbial fermentation is produced low temperature SOD end;
(6) with the fermented liquid 4,000～8 of (5), the centrifugal collection thalline of 000rpm is with centrifugal collecting precipitation after the distilled water wash several;
(7) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the SOD crude enzyme liquid;
(8) different with the use object according to different needs, the crude enzyme liquid that (7) are obtained further concentrates, separation and purification, is prepared into the low temperature SOD preparation of different activities, purity and formulation.
Embodiment three:
(1) medium preparation
1. strain activation and culture base: glucose 10.0g, Carnis Bovis seu Bubali cream 14.0g, peptone 7.0g, yeast powder 4.0g, agar 20.0g, pH 7.0, zero(ppm) water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
2. bacterial classification domestication by low temperature substratum: glucose 8.0～16.0g, Carnis Bovis seu Bubali cream 10.0～25.0g, peptone 3.0～12.0g, yeast powder 2.0～9.0g, agar 10.0～30.0g, zero(ppm) water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
3. liquid seed culture medium: glucose 5.0～15.0g, Carnis Bovis seu Bubali cream 8.0～20.0g, peptone 5.0～15.0g, yeast powder 2.0～10.0g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
4. enzymatic production substratum: glucose 10.0～30.0g, (NH
4)
2SO
43.0～10.0g, steeping water 5.0～15.0g, yeast powder 2.0～8.0g, CaCO
31.0～6.0g, MgSO
40.1～0.9g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
(2) will produce the bacterial classification of SOD, carry out initial activation by the bacterial strain explanation that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) bacterial classification that (2) activation is good domestication by low temperature step by step, acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃ → 16 ℃ → 14 ℃ → 12 ℃ → 10 ℃, makes its well-grown in low temperature environment from high to low;
(4) produce bacterium by the SOD of ordinary method after and cultivate 24～36h at 10～16 ℃ and then carry out enlarged culturing step by step, be prepared into liquid first order seed and secondary seed by 5～8% inoculum size with domestication by low temperature;
(5) inoculum size that the secondary seed that (4) is prepared is pressed fermentating liquid volume 7～9% inserts in the product enzyme substratum of 100L, and when cultivating 72～96h for 14～16 ℃, promptly microbial fermentation is produced low temperature SOD end;
(6) with the fermented liquid 4,000～8 of (5), the centrifugal collection thalline of 000rpm is with centrifugal collecting precipitation after the distilled water wash several;
(7) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the SOD crude enzyme liquid;
(8) different with the use object according to different needs, the crude enzyme liquid that (7) are obtained further concentrates, separation and purification, is prepared into the low temperature SOD preparation of different activities, purity and formulation.
Claims (3)
1. a microbial fermentation is produced the method for low temperature superoxide-dismutase, may further comprise the steps:
The mikrobe that (1) will produce superoxide-dismutase is domestication by low temperature step by step, and acclimation temperature 25 ℃ → 22 ℃ → 20 ℃ → 18 ℃ → 16 ℃ → 14 ℃ → 12 ℃ → 10 ℃, makes its well-grown under low temperature environment from high to low;
(2) produce bacterium 10～16 ℃ of enlarged culturing step by step by the superoxide-dismutase of ordinary method after, be prepared into liquid first order seed and secondary seed domestication by low temperature;
(3) with liquid first order seed or secondary seed, 3～9% inoculum sizes of pressing fermentating liquid volume insert in the liquid fermentation medium, and when cultivating 72～144h for 10～16 ℃, promptly microbial fermentation is produced the end of low temperature superoxide-dismutase;
(4) with the fermented liquid 4,000～8 of (3), the centrifugal collection thalline of 000rpm is with centrifugal collecting precipitation after the distilled water wash several;
(5) deposition is suspended in the damping fluid, adds silica sand and grind fragmentation, 10,000～14,000rpm is centrifugal, and the supernatant of collecting is the superoxide-dismutase crude enzyme liquid;
(6) according to different needs with to use object different, can also the crude enzyme liquid that (5) obtain is further concentrated, separation and purification, be prepared into the zymin of different activities, purity and formulation.
2. method according to claim 1 further comprises in step (6) afterwards: the crude enzyme liquid that step (6) is obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
3. method according to claim 1 and 2, wherein bacterial classification domestication by low temperature substratum, liquid seed culture medium, product enzyme substratum are respectively:
(1) bacterial classification domestication by low temperature substratum: glucose 8.0～16.0g, Carnis Bovis seu Bubali cream 10.0～25.0g, peptone 3.0～12.0g, yeast powder 2.0～9.0g, agar 10.0～30.0g, zero(ppm) water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
(2) liquid seed culture medium: glucose 5.0～15.0g, Carnis Bovis seu Bubali cream 8.0～20.0g, peptone 5.0～15.0g, yeast powder 2.0～10.0g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
(3) enzymatic production substratum: glucose 10.0～30.0g, (NH
4)
2SO
43.0～10.0g, steeping water 5.0～15.0g, yeast powder 2.0～8.0g, CaCO
31.0～6.0g, MgSO
40.1～0.9g, tap water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104164465A (en) * | 2014-07-14 | 2014-11-26 | 北京泰德制药股份有限公司 | Fermentation technology for expressing recombinant protein |
| CN105567650A (en) * | 2016-01-13 | 2016-05-11 | 广州市康优元生物科技有限公司 | Bacterial superoxide dismutase and preparing method thereof |
| CN107213029A (en) * | 2017-05-22 | 2017-09-29 | 大连大学 | A kind of moisturizing toner for adding low-temperature superoxide dismutase and preparation method thereof |
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| CN1807610A (en) * | 2006-01-23 | 2006-07-26 | 迟乃玉 | Method for producing low temperature cellulase using microbe fermentation |
| CN102093987A (en) * | 2009-12-11 | 2011-06-15 | 大连大学 | Method for producing low-temperature phytase by fermenting microorganisms |
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2012
- 2012-04-12 CN CN2012101067438A patent/CN102653733A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1807610A (en) * | 2006-01-23 | 2006-07-26 | 迟乃玉 | Method for producing low temperature cellulase using microbe fermentation |
| CN102093987A (en) * | 2009-12-11 | 2011-06-15 | 大连大学 | Method for producing low-temperature phytase by fermenting microorganisms |
Non-Patent Citations (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104164465A (en) * | 2014-07-14 | 2014-11-26 | 北京泰德制药股份有限公司 | Fermentation technology for expressing recombinant protein |
| CN105567650A (en) * | 2016-01-13 | 2016-05-11 | 广州市康优元生物科技有限公司 | Bacterial superoxide dismutase and preparing method thereof |
| CN107213029A (en) * | 2017-05-22 | 2017-09-29 | 大连大学 | A kind of moisturizing toner for adding low-temperature superoxide dismutase and preparation method thereof |
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Application publication date: 20120905 |