CN103045572B - Optimization of quambalaria cyanescens fermentation medium - Google Patents
Optimization of quambalaria cyanescens fermentation medium Download PDFInfo
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- CN103045572B CN103045572B CN201110397707.7A CN201110397707A CN103045572B CN 103045572 B CN103045572 B CN 103045572B CN 201110397707 A CN201110397707 A CN 201110397707A CN 103045572 B CN103045572 B CN 103045572B
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- rennin
- spore
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Abstract
The invention relates to an optimization of a quambalaria cyanescens fermentation medium after being subjected to mutation breeding. The concentration of the quambalaria cyanescens fermentation medium is optimized by changing a carbon source, a nitrogen source, an inorganic salt, a growth factor and the like of the fermentation medium through a single factor experiment, and an optimal fermentation culture medium formula is finally determined. The confirmatory experiments show that the chymosin enzyme activity of fermentation liquid can reach up to 310.36U/mL, so that the enzyme activity is improved by 182.15% than 110.00SU/mL of an initial enzyme activity.
Description
[technical field]
The mould fermention medium of blueness thorn spore that the present invention relates to mutagenic and breeding, belongs to biotechnology and enzyme preparation technical field.
[background technology]
It is the bacterial strain of our the strain producing lab ferment that separation and purification goes out from Red kojic rice that blueness is stung spore mould.But the rennin vigor of this strain bacterium is not high, it is mould as starting strain that we sting spore take blueness, application ultraviolet ray and ethyl sulfate composite factor mutagenesis starting strain.The excellent species DU10 (its microbial preservation number is: CGMCC 5423) of mutagenic and breeding, take glucose potato culture as fermention medium, the work of fermented liquid rennin enzyme is in 110SU/mL left and right fluctuation, and the institute's enzyme that produces work is not ideal enough.Therefore, still need the fermention medium of DU10 to be optimized.
Microbial growth, metabolism, to go down to posterity be all very complicated biological process, in these processes, need a large amount of nutritive substances as substrate or inductor, also there are some materials can microbial growth metabolism be produced and be suppressed, even the phenomenon on producing the impact of enzyme situation also likely appears changing along with the variation of concentration in same material simultaneously.The design of substratum can be from the viewpoint of carbon source, nitrogenous source, inorganic salt, somatomedin and precursor etc., and its concentration is optimized.
[summary of the invention]
The object of this invention is to provide the method for the mould strain excellent medium optimization of blue thorn spore after a kind of mutagenic and breeding, improve the mould fermented liquid rennin of blue thorn spore and live.Main technique route: utilize the blueness thorn mould strain excellent DU10 of spore (its microbial preservation number is: CGMCC 5423) after mutagenesis, live by changing culture medium prescription raising rennet ferment liquid enzyme.
The technical program is as follows:
1) aseptic technique is injected 10mL sterilized water to the DU10PDA inclined-plane of preserving, and washes the spore on lower inclined plane, and then vibration disperses spore, G
3glass funnel filters, and spore concentration is counted and adjusted to blood cell plate is 10
7～10
8individual/mL.
2), in fermentation basic medium, replace respectively carbon source, nitrogenous source wherein, research carbon and nitrogen sources and the impact of concentration on fermented liquid quality thereof.Culture condition is: inoculation prepared by inclined-plane 10
7～10
8individual/mL spore suspension, 1%, 30 ℃ of inoculum size, 150r/min, shaking culture 96h.Rennin enzyme (MCA) and the proteolytic activity (PA) alive of measuring fermented liquid, fermented liquid rennin is lived higher, and MCA/PA ratio is larger, and the institute's producing lab ferment that ferments is better.
3) select step 2) definite suitable carbon and nitrogen sources and concentration thereof; Add different metal ion and surfactant emulsion agent, investigate their impacts on enzymatic production quality, determine metal ion that Optimal Medium adds and kind and the concentration of surfactant emulsion agent.Culture condition and requirement are with step 2).
4) checking of best medium formula
Fermentor tank checking: according to the best medium of experiment gained, preparation 4L fermention medium packs 10L fermentor tank into, and culture condition is with step 2), survey fermented liquid rennin enzyme and live.
Above-mentioned steps 2) in fermentation basic medium formula as follows, be percentage composition:
Glucose 2%, peptone 0.4%, yeast extract paste 0.1%, distilled water is supplied 1000mL, pH nature, 121 ℃ of sterilizing 20min.
Mentioned reagent and culture medium prescription, if no special instructions, be the conventional reagent in this area and substratum.
Above-mentioned steps 2) in the measuring method of rennin vigor be: with the CaCl of 0.01mol/L
2the skimming milk of preparation 100g/L, places 40min formed cow's milk system is tended towards stability, and then gets 5mL skimming milk 36 ℃ of insulation 5min in test tube.The centrifugal 10min of fermented liquid 5000r/min makes mycelia precipitation, getting its supernatant liquor 0.5mL adds in the skimming milk of insulation, on eddy mixer, mix rapidly and start timing, when there being the fritter of condensing on test tube wall, accurate recording fermented supernatant fluid makes the time of Coagulation of milk.The required rennin amount of skimming milk 1mL that definition 40min solidifies 100g/L is a Soxhlet unit (Soxhelt unit, SU).
Rennin formula alive is:
In formula: T-curdled milk time/s; D-extension rate.
Above-mentioned steps 2) mensuration of proteolytic activity adopts Folin reagent method, uses 10mL0.1NNaOH solubilising casein, and pH6.2 phosphorus damping fluid is settled to 100mL.36 ℃ of insulations of fermented liquid, 660nm place colorimetric is surveyed enzyme activity.Defining the enzyme amount that 36 ℃, the every 1min caseinhydrolysate of pH6.2 produce 1 μ g tyrosine is a protease activity unit of force (U).
Proteolytic activity formula is:
In formula: the tyrosine content μ g/mL of OD value correspondence on typical curve when A-protease activity is measured.
Tyrosine typical curve: different concns tyrosine 1mL, Folin reagent 1mL and 0.4mol/LNa
2cO
35mL mixes, and 36 ℃ of water-bath colour developing 20min, survey the OD of 660nm place value, draw the typical curve of different concns tyrosine and OD value
Beneficial effect of the present invention:
1. blueness stings spore mould to be a kind of novel rennet producing strain, its substratum is optimized and has important Research Significance;
2. by the mould medium optimization of blueness thorn spore to mutagenesis, improved fermented liquid rennin vigor.
[biomaterial preservation information]
Bacterial classification DU10, Classification And Nomenclature is blue thorn spore mould (Sporothrix Cyanescens), be preserved in common micro-organisms culture presevation administrative center of China Committee for Culture Collection of Microorganisms, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preserving number is CGMCC 5423, and preservation date is on November 3rd, 2011.
[embodiment]
Below in conjunction with embodiment, method of the present invention is described further, but is not limited to this.It is our separation and purification goes out from Red kojic rice strain producing lab ferment bacterial strain that the blueness using in embodiment is stung spore mould.We are just sifted out rennin superior strain, and then are carried out multiple sieve by ultraviolet ray, ethyl sulfate composite factor mutagenic treatment starting strain from bacterial strain after treatment.Finally the bacterial strain sifting out is again carried out the analysis of genetic stability and enzymatic production quality.Finally select strain excellent DU10 (microbial preservation number is: CGMCC 5423), the present invention, by changing culture medium prescription, has improved fermented liquid rennin and has lived.
Embodiment 1
1) different carbon sources and carbon source concentration are on producing the impact of enzyme
Carbon source can provide the energy for microorganism growth breeding, is again the synthetic necessary composition of thalline, also for object product provides required carbon component.There is difference in the enzyme system that microorganism self exists, utilizes different carbon metabolisms also to answer different on the impact of fermented liquid quality.Carbon source in fermention medium is replaced with glucose, fructose, sucrose, lactose and Zulkovsky starch successively.
Carbon source kind has difference to the quality impact of fermented liquid: the rennin enzyme that glucose is carbon source is lived maximum, and MCA/PA is also larger; Rennin enzyme take lactose and Zulkovsky starch as carbon source live and MCA/PA ratio all very little, therefore selection glucose is as the carbon source of the fermention medium of rennin superior strain.In the time that the concentration of glucose in substratum is 3.3～3.8%, rennin enzyme is lived higher, and MCA/PA ratio is also larger, so while utilizing rennin superior strain enzymatic production, glucose concn used is 3.3～3.8%.
2) different nitrogen sources on and nitrogen concentration on producing the impact of enzyme
Nitrogenous source is the source that forms cell protein and nucleic acid substances, has important relationship with thalline eubolism.In fermention medium, nitrogenous source composition is used peptone, extractum carnis, urea, NaNO successively
3(NH
4)
2sO
4replace the impact of research different nitrogen sources on enzymatic production.Peptone is during as the nitrogenous source of rennet producing strain, and enzymatic production has good performance, and this may be easy to induction with the nitrogen in peptone, and to produce enzyme relevant, and the nitrogenous source of fermention medium is elected peptone as.Adjust the concentration of peptone in fermention medium, fermentation culture is measured the rennin enzyme of fermented liquid and is lived, and in the time that in substratum, peptone concentration is 0.38～0.42%, fermented liquid rennin is lived higher, and MCA/PA ratio is also larger.Therefore,, while utilizing rennin superior strain fermentation culture, the peptone concentration of fermenting used is 0.38～0.42%.
3) yeast extract paste concentration is on producing the impact of enzyme
Yeast extract paste both can be used as culture media nitrogen source, can be again microbial reproduction required somatomedin is provided.In fermention medium, increase yeast extract paste concentration, the impact of research different concns yeast extract paste on fermented liquid quality.When yeast extract paste concentration is 0.58～0.62%, rennin enzyme is lived higher, and MCA/PA ratio is also larger, and too high too low yeast extract paste concentration all can affect the output of growth and breeding and the rennin of DU10.Therefore, the suitable concn of yeast extract paste is 0.58～0.62%.
4) selection of metal ion and surfactant
Inorganic salt contain microorganism growth and breed necessary metal ion, and these metal ions can be divided into macroelement and trace element according to microorganism to their demand, macroelement mainly contains P, S, K, Mg, Ca, Na and Fe etc., trace element has Cu, Zn, Mn, Mo and Co etc., and trace element is because demand is very little general enough in some composition of substratum.Select concentration K
2hPO
4, MgSO
4, FeSO
4, MnSO
4, CaCl
2, NaCl, probe into the impact of different metal ion pair inulinase-producing activity.Emulsifying agent is conducive to the dispersion of each composition in substratum, also can make microbial profile more even.Selecting tween-80 is surfactant emulsion agent, investigates the impact of this emulsifying agent on inulinase-producing activity.
In fermention medium, add respectively 0.001% MnSO
4, other inorganic salt of 0.5% and 0.5% surfactant, do control experiment simultaneously.Result shows, K
2hPO
4, MgSO
4, FeSO
4with NaCl, the quality of enzymatic production is not had to promoter action, and MnSO
4, tween-80, CaCl
2can obviously promote strain fermentation yield of enzyme.Therefore choose MnSO
4, tween-80, CaCl
2do different concns to producing the test of enzyme impact.
Example 2
1) aseptic technique is injected 10mL sterilized water to the DU10PDA inclined-plane of preserving, and washes the spore on lower inclined plane, and then vibration disperses spore, G
3glass funnel filters, and spore concentration is counted and adjusted to blood cell plate is 10
7～10
8individual/mL.
2) in the 250mL triangular flask that contains 50mL substratum, inoculation contains spore 10
7～10
8the spore suspension of individual/mL, 30 ℃, 150r/min, shaking culture 96h.
Fermention medium component is as follows, is all weight percentage:
Glucose 3.3%; Peptone 0.4%; Yeast extract paste 0.6%; Calcium chloride 0.05%; MnSO
40.25%; Tween-80 0.1%, supplies distilled water 1000mL, pH nature.
3) the rennin enzyme of measuring fermented liquid is lived and proteolytic activity, and fermented liquid MCA and MCA/PA ratio are larger, and the institute's producing lab ferment that ferments is better.The rennin work that records fermented liquid is 288.21SU/mL, and than original strain rennin, 110.00SU/mL alive has improved 162.01%,
Example 3
1) aseptic technique is injected 10mL sterilized water to the DU10PDA inclined-plane of preserving, and washes the spore on lower inclined plane, and then vibration disperses spore, G
3glass funnel filters, and spore concentration is counted and adjusted to blood cell plate is 10
7～10
8individual/mL.
2) in the 250mL triangular flask that contains 50mL substratum, inoculation contains spore 10
7～10
8the spore suspension of individual/mL, 28～32 ℃, 150～160r/min, shaking culture 96～120h.
Fermention medium component is as follows, is all weight percentage;
Glucose 3.6%; Peptone 0.4%; Yeast extract paste 0.6%; Calcium chloride 0.05%; MnSO
40.25%; Tween-80 0.1%, supplies distilled water 1000mL, pH nature.
3) the rennin enzyme of measuring fermented liquid is lived and proteolytic activity, and fermented liquid MCA and MCA/PA ratio are larger, and the institute's producing lab ferment that ferments is better.The rennin work that records fermented liquid is 296.58SU/mL, and than original strain rennin, 110.00SU/mL alive has improved 169.62%.
When understanding, above-mentioned embodiment is only illustrative explanation, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (3)
1. the application of substratum in the fermentation of blueness thorn spore trichoderma strain, the preserving number of described blueness thorn spore trichoderma strain is: CGMCC5423, described substratum comprises the glucose 3.3～3.8% of weight percent; Peptone 0.38～0.42%; Yeast extract paste 0.58～0.62%; Calcium chloride 0.05%; MnSO
40.20～0.25%; Tween-80 0.10～0.12%.
2. application according to claim 1, the solvent that it is characterized in that substratum is water.
3. application according to claim 1, the solvent that it is characterized in that substratum is distilled water or pure water.
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Citations (2)
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|---|---|---|---|---|
| CN101503659A (en) * | 2009-03-11 | 2009-08-12 | 中国农业大学 | Trichoderma harzianum strain and use thereof |
| CN101748082A (en) * | 2008-12-11 | 2010-06-23 | 吉林省农业科学院 | Lactobacillus leavening agent, preparation method thereof and special bacterial strain |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748082A (en) * | 2008-12-11 | 2010-06-23 | 吉林省农业科学院 | Lactobacillus leavening agent, preparation method thereof and special bacterial strain |
| CN101503659A (en) * | 2009-03-11 | 2009-08-12 | 中国农业大学 | Trichoderma harzianum strain and use thereof |
Non-Patent Citations (3)
| Title |
|---|
| 王明强，张惟广.发酵法生产凝乳酶的概述.《四川食品与发酵》.2008,第44卷(第2期),第21-24页. * |
| 红曲米中产凝乳酶菌株的分离筛选与特性的研究;顾振磊等;《中国酿造》;20091231(第10期);第58-60页 * |
| 顾振磊等.红曲米中产凝乳酶菌株的分离筛选与特性的研究.《中国酿造》.2009,(第10期),第58-60页. |
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| CB03 | Change of inventor or designer information |
Inventor after: Wang Chengzhong Inventor after: Yuan Yaguang Inventor after: Zhang Zhiguo Inventor after: Zhao Xiaohong Inventor before: Wang Chengzhong Inventor before: Zhang Zhiguo Inventor before: Sun Qiuyan |
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Free format text: CORRECT: INVENTOR; FROM: WANG CHENGZHONG ZHANG ZHIGUO SUN QIUYAN TO: WANG CHENGZHONG YUAN YAGUANG ZHANG ZHIGUO ZHAO XIAOHONG |
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