CN103232983B - Culture medium for producing amylase by using deep marine geobacillus sp (Geobacillus sp.) - Google Patents
Culture medium for producing amylase by using deep marine geobacillus sp (Geobacillus sp.) Download PDFInfo
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Abstract
The invention relates to a culture medium for producing amylase by using deep marine geobacillus sp (Geobacillus sp.). The culture medium is suitable for the growth of the deep marine geobacillus sp (Geobacillus sp.) and can greatly increase the output of the amylases.
Description
Technical field
The invention belongs to biological technical field, more specifically, the present invention relates to and produce diastatic substratum and preparation method thereof for ocean, deep-sea thermophilic bacterium Geobacillus sp. (being more preferably Geobacillus sp.4j).
Background technology
Thermophilic marine microorganism is the class thermophilic microorganism resource deriving from the particular surroundingss such as underground heat oil-containing geological stratification, deep-sea volcanic mud, deep-sea hydrothermal port.Thermophilic microorganism enjoys people to pay close attention to because of it to the adaptability of high temperature uniqueness, generally refers to that optimum growth temperature is greater than a quasi-microorganism of 45 DEG C.As thermophilic microorganism, it one of is mainly applied is the Application and Development of Zimadzhunt L 340.Zimadzhunt L 340 gets most of the attention because of the high advantage of its thermostability, and wherein the most famous is exactly discovery and the exploitation of Tag enzyme, and making PCR react this revolutionary technical development becomes one of of paramount importance technology of life science.The enzyme tool high thermal stability separated from thermophile bacteria known at present, rate of mass transfer are high, chemically-resistant denaturing agent, be easy to the features such as purifying.
Ocean, deep-sea thermophilic bacterium Geobacillus sp.4j, by being be separated first by Chinese Sea the 3rd institute to obtain, derive from deep-sea, optimum growth temperature at a kind of moderate thermophilic microorganism of 60 DEG C.Because it derives from this extreme environment of deep-sea, special physiology may be had to envrionment conditionss such as dissolved oxygen, high salt, high temperature, high pressure corresponding.In application aspect, Geobacillus sp.4j finds the amylase that can produce degraded starch at present.Can this kind of diastatic activity and thermostability etc. need to be explored further, and be used for the also worth research of a kind of amylase of suitability for industrialized production.
But it is very low that thermophile bacteria Geobacillus sp.4j grows diastatic output in original culture medium, become the bottleneck of every research, produce the field of enzyme especially in its mass-producing fermentation culture.If diastatic output can not be improved, be just difficult to the exploration carrying out corresponding enzymology and suitability for industrialized production.
Therefore, by optimizing its culture environment, improve its zymotechnique and become the task of top priority.Substratum is the matrix of microorganism growth, develops a kind ofly to produce for ocean, deep-sea thermophilic bacterium Geobacillus sp.4j the important foundation that diastatic novel culture medium is at present work.
Summary of the invention
The object of the present invention is to provide and produce diastatic substratum, its preparation method and application for ocean, deep-sea thermophilic bacterium Geobacillus sp..
In a first aspect of the present invention, provide the substratum of ocean, a kind of deep-sea thermophilic bacterium Geobacillus sp., described substratum comprises following component:
In a preference, described ocean, deep-sea thermophilic bacterium Geobacillus sp. is Geobacillussp.4j.
In another preference, described substratum comprises following component:
In another preference, described substratum comprises following component:
Wherein, each component content in the medium can float in ± 10% (more preferably ± 5%) scope.
In another preference, in described substratum, the trace element also containing significant quantity (being suitable for the amount of bacterial growth); Preferably, micronutrient levels is:
In another preference, in described substratum, each component is formulated in water (preferably, described water is deionized water); And/or the pH value 6 ± 0.5 of described substratum.
In another aspect of this invention, providing the purposes of arbitrary described substratum above, for cultivating ocean, deep-sea thermophilic bacterium Geobacillus sp., producing amylase.
In another aspect of this invention, provide a kind of method preparing the substratum of arbitrary described ocean, deep-sea thermophilic bacterium Geobacillus sp. above, described method comprises:
A described Zulkovsky starch is carried out gelatinization by (), cooling;
B () is by described yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate mixing;
C the solution that above-mentioned two steps obtain mixes by (), interpolation nickel chloride solution and optionally trace element;
Wherein, the consumption of each component is according to described in arbitrary above.
In another preference, in step (a), described gelatinization, for described Zulkovsky starch being made suspension liquid with a small amount of water (being preferably deionized water), is then slowly poured 90-100 DEG C of deionized water into and constantly stirs, making translucent liquid.
In another preference, after step (c), comprise further: pH is adjusted to 6 ± 0.5, at 121 DEG C of sterilizing 20-30min.
In another aspect of this invention, one is provided to utilize ocean, deep-sea thermophilic bacterium Geobacillus sp. to produce diastatic method, described method comprises: utilize arbitrary described ocean, culture medium culturing deep-sea thermophilic bacterium Geobacillus sp. above, thus produce amylase.
In a preference, the inoculum size of ocean, deep-sea thermophilic bacterium Geobacillus sp. 1-5% is by volume inoculated into arbitrary described substratum above, 60 DEG C, static condition bottom fermentation cultivation 72-96h, carry out an oscillation treatment every 12h in quiescent culture process, the mixing of bacterium liquid will be cultivated; After cultivating 72h and 96h, centrifugation fermented supernatant fluid, wherein containing amylase.
In another preference, for the OD of seed liquor inoculated
600for 1.8-2.4, colony number density is about 2.1 × 10
6-2.8 × 10
6cFU/ml.
In another aspect of this invention, provide a kind of test kit for cultivating ocean, deep-sea thermophilic bacterium Geobacillus sp., described test kit comprises: arbitrary described substratum above.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Embodiment
The present inventor, through long-term and deep research, has researched and developed a kind of cultivation ocean, deep-sea thermophilic bacterium Geobacillus sp. (being more preferably Geobacillus sp.4j) that is suitable for newly and has made it the diastatic culture medium prescription of High-efficient Production.Substratum of the present invention, is suitable for the growth of ocean, deep-sea thermophilic bacterium Geobacillus sp., and can improve diastatic output widely.
As used herein, described " containing ", " having " or " comprising " include " comprising ", " primarily of ... form ", " substantially by ... form " and " by ... form "; " primarily of ... form ", " substantially by ... form " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".
Term " substratum of ocean, deep-sea of the present invention thermophilic bacterium Geobacillus sp. " refers to the composition containing Zulkovsky starch, yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate, nickelous chloride and optional trace element; Or the composition be substantially made up of Zulkovsky starch, yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate, nickelous chloride and optional trace element.In the composition, described Zulkovsky starch, yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate, nickelous chloride and optional trace element account for the 80-100% of substratum gross weight, preferably account for 90-100%, more preferably account for 95-100%, as 98%, 99%.
As optimal way of the present invention, the consumption for each component preparing ocean, deep-sea of the present invention thermophilic bacterium Geobacillussp. (being more preferably Geobacillus sp.4j) substratum is as shown in table 1.
Table 1
As optimal way of the present invention, also add trace element in described substratum, the addition of trace element is advisable to meet needed for thalli growth and production, and those skilled in the art can empirically add.As optimal way of the present invention, the consumption of micro-each component is as shown in table 2.
Table 2
After learning the component that ocean, deep-sea of the present invention thermophilic bacterium Geobacillus sp. (being more preferably Geobacillussp.4j) substratum is used and formula thereof, those skilled in the art can prepare easily.
The component (raw material) of the application of substratum of the present invention is business-like general chemistry product, price comparison is low, prepare also very simple, compared with the substratum of prior art, novel culture medium of the present invention significantly can promote ocean, deep-sea thermophilic bacterium Geobacillus sp. fermentative production amylase, diastatic output is significantly improved, and is conducive to large-scale industrial production.
As optimal way of the present invention, when ocean, deep-sea thermophilic bacterium Geobacillus sp. (being the more preferably Geobacillus sp.4j) substratum described in preparing, first accurately take each component respectively, they are mixed in water.Described water is preferably deionized water.Preferred, a kind of method for the preparation of above-mentioned substratum is provided, comprises step:
A described Zulkovsky starch is carried out gelatinization by (), cooling;
B () is by described yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate mixing;
C the solution that above-mentioned two steps obtain mixes by (), interpolation nickel chloride solution and optionally trace element.
Described Zulkovsky starch first need carry out gelatinization process, and object is the problem preventing from luming in sterilization process.Any method that the gelatinization of Zulkovsky starch can adopt those skilled in the art known.As optimal way of the present invention, described gelatinization, for described Zulkovsky starch being made suspension liquid with a small amount of water (being preferably deionized water), is then slowly poured 90-100 DEG C of deionized water into and constantly stirs, making translucent liquid.
As optimal way of the present invention, after step (c), comprise further: pH is adjusted to 6 ± 0.5, at 121 DEG C of sterilizing 20-30min.
Present invention also offers and use ocean, described culture medium culturing deep-sea thermophilic bacterium Geobacillus sp. (being more preferably Geobacillus sp.4j) the diastatic fermentation process of fermentative production, comprise step: ocean, the culture medium culturing deep-sea thermophilic bacterium Geobacillus sp. described in utilization, thus produce amylase.
Present invention also offers the diastatic detection method that a kind of above-mentioned fermentation process is produced, its operation steps comprises:
A. with rifle head picking list bacterium colony from the solid plate of fresh Geobacillus sp.1j of inoculating needle or sterilizing, be seeded in the 250ml shaking flask that 80ml activated seed substratum is housed, 60 DEG C, 100rpm shaker fermentation cultivates 16-20h, obtains fresh seeds liquid;
B. described fresh seeds liquid is got, be equipped with in the 250ml shaking flask of novel culture medium described in 50ml by the inoculum size access of 1% (v/v), silica gel plug seals, 60 DEG C, be statically placed in fermentation culture 72-96h in high temperature constant temperature shaking table, for whole quiescent culture process, a 15min is carried out every 12h, the vibration of 100rpm, will cultivate the mixing of bacterium liquid, bacterium liquid is mixed.After cultivating 72h and 96h, 12000rpm, 4 DEG C of centrifugal 3min, obtain fermented supernatant fluid and be used for enzyme mensuration alive, get wherein larger value.
Preferably, after the bacterial classification that-80 DEG C are preserved is thawed, 1 ring bacterium liquid is got with aseptic inoculation ring, solid plate activation medium is rule, be placed in 60 DEG C of constant incubators, activation culture 8-12h, obtain fresh solid dull and stereotyped, then be placed in 4 DEG C of refrigerators stand-by, later solid plate reactivated once every 30 days.
Preferably, often liter of activation medium includes yeast powder 2.5g, peptone 2.5g, nitrilotriacetic acid(NTA) 0.1g, terra alba 0.04g, magnesium chloride 0.2g, ironic citrate 0.01675g, liquid microelement I0.5ml, phosphate buffer 1 00ml, all the other are deionized water, and pH is adjusted to about 7.35, at 121 DEG C of sterilizing 20-30min.
Preferably, often liter of solid plate activation medium adds agar powder 15-20g on the basis of activation medium.
Preferably, the OD of fresh seed liquor fresh seeds liquid during inoculation
600for 1.8-2.4, colony number density is about 2.1 × 10
6-2.8 × 10
6cFU/ml.
Preferably, containing dipotassium hydrogen phosphate 5.44g, disodium hydrogen phosphate dodecahydrate 43g in often liter of phosphate buffered saline buffer, all the other are deionized water, and pH is adjusted to about 7.2.
Medium component of the present invention is clear and definite, addition is low, with low cost, preparation manipulation is convenient, ocean, deep-sea thermophilic bacterium Geobacillus sp.4j can be significantly improved and grow the heat-resisting diastatic output of production in high temperature environments, make the diastatic enzyme work in fermented supernatant fluid bring up to 6.36U/ml by original 2U/ml.This fermentation research for ocean, deep-sea thermophilic bacterium product enzyme and novel diastatic exploitation have great importance.
Present invention also offers a kind of diastatic detection method utilizing described fermentation process to obtain, comprise step: get some test tubes mark, draw the starch substrates (pH7.50) of appropriate 1% with pipettor, put into 60 DEG C of water bath with thermostatic control preheating 10min.Then the fermented supernatant fluid that appropriate described fermentation process obtains is added in test tube, mix immediately, accurately react for some time.After reaction, reaction tube is transferred in ice-water bath immediately, add appropriate amount of deionized water and appropriate DNS reagent immediately, mix.Then after being transferred to 100 DEG C of accurate clock reaction 5min of water bath with thermostatic control, put into mixture of ice and water immediately to cool, then add certain volume deionized water, the absorbance A at spectrophotometric determination 540nm place is used in mixing again, according to the reducing sugar content in light absorption value A conversion response liquid.Then the reducing sugar content natively had in Simultaneously test starch substrates and fermented supernatant fluid, can calculate diastatic enzyme in described fermented supernatant fluid live by calculating corresponding difference.Wherein 1 enzyme unit definition alive is the enzyme amount that under above-mentioned condition, per minute catalytic decomposition produces 1 micromole's reducing sugar.
Beneficial effect of the present invention is specific as follows:
1. novel culture medium composition of the present invention comprises the common agents such as Zulkovsky starch, yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate, liquid microelement addition is low, raw material is easy to get, and consumption is few, and cost is low;
2., when preparing novel culture medium of the present invention, first starch is spent ionized water and make suspension, boil gelatinization, then cool, by all the other components dissolved in deionized water, both mixing, then add a small amount of liquid microelement, constant volume, preparation is simple;
3. adopt novel culture medium of the present invention to cultivate ocean, deep-sea thermophilic bacterium Geobacillus sp.4j, this bacterial strain grows diastatic output and is significantly increased in this novel culture medium, makes the diastatic enzyme work in fermented supernatant fluid bring up to 6.36U/ml by original 2U/ml.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
In addition, adopt the equipment such as the high temperature constant temperature shaking table of different manufacturers, may cause a little difference of experimental result, difference like this belongs to normal phenomenon.
Embodiment 1, substratum 1
1) Zulkovsky starch gelatinization
Take 10g Zulkovsky starch, make suspension liquid with 20ml deionized water, then slowly pour in the deionized water just boiled and also constantly stir, make translucent uniform viscous liquid, cooling.
2) substratum preparation
Take yeast powder 4g, SODIUMNITRATE 3g, sodium-chlor 20g, anhydrous magnesium sulfate 1.67g, magnesium chloride 8.92g, Repone K 0.33g, calcium chloride 0.1g, dipotassium hydrogen phosphate 0.183g, ferrous ammonium sulphate 0.01g, pour appropriate amount of deionized water into mentioned component is fully dissolved, pour step 2 into again) the starch pasting liquid of cooling prepared, adds liquid microelement I10ml, nickel chloride solution (10g/L) 50 μ L, 1000ml is settled to, pH regulator to 6 again with deionized water.
Wherein prepare liquid microelement I to prepare as follows:
Nitrilotriacetic acid(NTA) 12.8g, Iron dichloride tetrahydrate 1g, four chloride hydrate manganese 0.5g, four hydrated cobalt chloride 0.3g, Copper dichloride dihydrate 0.05g, Sodium orthomolybdate 0.05g, boric acid 0.02g, Nickel dichloride hexahydrate 0.02g, all the other are deionized water, are settled to 1000ml, and pH is adjusted to 6.8.Long-time storage needs the syringe-driven filter filtration sterilization of use 0.22 μm.
3) fermentation culture process
Ocean, deep-sea thermophilic bacterium Geobacillus sp.4j is provided by Chinese Sea the 3rd institute.Picking list bacterium colony from activated good solid plate, is seeded in the 250ml shaking flask that 80ml activated seed substratum is housed, and 60 DEG C, 100rpm shaker fermentation cultivation 18h, obtain fresh seeds liquid; The OD of fresh seeds liquid
600be that 2.1 (colony number density is about 2.4 × 10
6cFU/ml), by the inoculum size of 1% (v/v), the access of fresh seeds liquid is equipped with in the 250ml shaking flask of novel culture medium described in 50ml, 60 DEG C, be statically placed in fermentation culture 96h in high temperature constant temperature shaking table, for whole quiescent culture process, carry out the vibration of 15min, a 100rpm every 12h, the mixing of bacterium liquid will be cultivated, bacterium liquid is mixed.After cultivating 96h, 12000rpm, 4 DEG C of centrifugal 3min, obtain fermented supernatant fluid and be used for enzyme mensuration alive.
Wherein, often liter of activation medium includes yeast powder 2.5g, peptone 2.5g, nitrilotriacetic acid(NTA) 0.1g, terra alba 0.04g, magnesium chloride 0.2g, ironic citrate 0.01675g, liquid microelement I0.5ml, phosphate buffer 1 00ml, all the other are deionized water, and pH is adjusted to about 7.35, at 121 DEG C of sterilizing 20-30min.Containing dipotassium hydrogen phosphate 5.44g, disodium hydrogen phosphate dodecahydrate 43g in often liter of phosphate buffered saline buffer, all the other are deionized water, and pH is adjusted to about 7.2.
4) diastatic enzyme activity determination
Get some test tubes mark, draw starch substrates (adopting Sodium phosphate dibasic-citrate buffer solution gelatinization and the preparation of 0.05M, pH7.50) the 280 μ L of 10g/L gelatinization in advance, put into 60 DEG C of water bath with thermostatic control preheating 10min.Then fermented supernatant fluid 20 μ L is added in test tube, mix immediately, accurately react 10min.After reaction, reaction tube is transferred in ice-water bath immediately, add the DNS reagent of 200 μ L deionized waters and 300 μ L immediately, mix.Then after being transferred to 100 DEG C of accurate clock reaction 5min of water bath with thermostatic control, put into mixture of ice and water immediately to cool, then add ionized water 6ml, the absorbance A at spectrophotometric determination 540nm place is used in mixing again, according to the reducing sugar content in light absorption value A conversion response liquid.Then the reducing sugar content natively had in Simultaneously test starch substrates and fermented supernatant fluid, can calculate the diastatic enzyme that the content that produces reducing sugar with per minute hydrolysis characterizes and live, measurement result, enzyme is lived as 4.4U/ml.
Embodiment 2, substratum 2
1) Zulkovsky starch gelatinization
10g Zulkovsky starch 20ml deionized water is made suspension liquid, then slowly pours in the deionized water just boiled and also constantly stir, make translucent uniform viscous liquid, cooling.
2) substratum preparation
Take yeast powder 8g, SODIUMNITRATE 1g, sodium-chlor 20g, anhydrous magnesium sulfate 1.67g, magnesium chloride 15g, Repone K 0.33g, calcium chloride 0.15g, dipotassium hydrogen phosphate 0.183g, ferrous ammonium sulphate 0.01g, pour appropriate amount of deionized water into mentioned component is fully dissolved, pour step 1 into again) the starch pasting liquid of cooling prepared, adds liquid microelement I10ml, nickel chloride solution (10g/L) 50 μ L, 1000ml is settled to, pH regulator to 6 again with deionized water.
3) fermentation culture process
Step 3 with embodiment 1).
4) fermentation culture measures
Step 4 with embodiment 1), measure diastatic enzyme and live as 4.98U/ml.
Embodiment 3, substratum 3
1) Zulkovsky starch gelatinization
Take 9g Zulkovsky starch 20ml deionized water and make suspension liquid, then slowly pour in the deionized water just boiled and also constantly stir, make translucent uniform viscous liquid, cooling.
2) substratum preparation
Take yeast powder 8g, SODIUMNITRATE 7.5g, sodium-chlor 20g, anhydrous magnesium sulfate 1.67g, magnesium chloride 6g, Repone K 0.33g, calcium chloride 0.6g, dipotassium hydrogen phosphate 0.2g, ferrous ammonium sulphate 0.01g, pour appropriate amount of deionized water into mentioned component is fully dissolved, pour step 1 into again) the starch pasting liquid of cooling prepared, adds liquid microelement I12ml, nickel chloride solution (10g/L) 50 μ L, 1000ml is settled to, pH regulator to 6 again with deionized water.
3) fermentation culture process
Step 3 with embodiment 1).
4) fermentation culture measures
Step 4 with embodiment 1), measure diastatic enzyme and live as 5.08U/ml.
Embodiment 4, substratum 4
1) Zulkovsky starch gelatinization
Take 6g Zulkovsky starch 15ml deionized water and make suspension liquid, then slowly pour in the deionized water just boiled and also constantly stir, make translucent uniform viscous liquid.
2) substratum preparation
Take yeast powder 8g, SODIUMNITRATE 5g, sodium-chlor 15g, anhydrous magnesium sulfate 1.67g, magnesium chloride 2g, Repone K 0.33g, calcium chloride 0.6g, dipotassium hydrogen phosphate 0.4g, ferrous ammonium sulphate 0.01g, pour appropriate amount of deionized water into mentioned component is fully dissolved, pour step 1 into again) the starch pasting liquid of cooling prepared, adds liquid microelement I10ml, nickel chloride solution (10g/L) 50 μ L, 1000ml is settled to, pH regulator to 6 again with deionized water.
3) fermentation culture process
For ease of comparing, the step 3 with embodiment 1).
4) cell density measures
Step 4 with embodiment 1), measure diastatic enzyme and live as 6.2U/ml.
Embodiment 5, substratum 5
1) Zulkovsky starch gelatinization
Take 6g Zulkovsky starch 12ml deionized water and make suspension liquid, then slowly pour in the deionized water just boiled and also constantly stir, to becoming translucent uniform viscous liquid, cooling.
2) substratum preparation
Take yeast powder 8g, SODIUMNITRATE 5g, sodium-chlor 12.5g, anhydrous magnesium sulfate 1.67g, magnesium chloride 0.32g, Repone K 0.33g, calcium chloride 0.6g, dipotassium hydrogen phosphate 0.484g, ferrous ammonium sulphate 0.01g, pour appropriate amount of deionized water into mentioned component is fully dissolved, pour step 1 into again) the starch pasting liquid of cooling prepared, adds liquid microelement I10ml, nickel chloride solution (10g/L) 50 μ L, 1000ml is settled to, pH regulator to 6 again with deionized water.
3) fermentation culture process
Step 3 with embodiment 1).
4) cell density measures
Step 4 with embodiment 1), measure diastatic enzyme and live as 6.36U/ml.
Embodiment 6, control group example
1) Zulkovsky starch gelatinization
Take 30g Zulkovsky starch 50ml deionized water and make suspension liquid, then slowly pour in the deionized water just boiled and also constantly stir, to becoming translucent uniform viscous liquid, cooling.
2) substratum preparation
Take yeast powder 5g, peptone 5g, sodium-chlor 30g, anhydrous magnesium sulfate 1.67g, magnesium chloride 8.92g, Repone K 0.33g, calcium chloride 0.1g, dipotassium hydrogen phosphate 0.183g, ferrous ammonium sulphate 0.01g, pour appropriate amount of deionized water into mentioned component is fully dissolved, pour step 1 into again) the starch pasting liquid of cooling prepared, adds liquid microelement I10ml, nickel chloride solution (10g/L) 50 μ L, 1000ml is settled to, pH regulator to 7.35 again with deionized water.
3) fermentation culture process
Step 3 with embodiment 1).
4) cell density measures
Step 4 with embodiment 1), measure diastatic enzyme and live as 2U/ml.
Can find from embodiment 1 ~ 6: utilize novel culture medium of the present invention and preparation method, make the diastatic enzyme work in the diastatic fermented supernatant fluid of ocean, deep-sea thermophilic bacterium Geobacillus sp.4j production hot temperature degree bring up to more than 4U/ml by 2U/ml in the past, more can reach 6.36U/ml.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (8)
1. ocean, deep-sea thermophilic bacterium (
geobacillus sp.) substratum of 4j, it is characterized in that, described substratum comprises following component:
In described substratum, the trace element also containing significant quantity; Micronutrient levels is:
2. substratum as claimed in claim 1, it is characterized in that, described substratum comprises following component:
Wherein, each component content in the medium can float in ± 10% scope.
3. the substratum as described in as arbitrary in claim 1-2, it is characterized in that, in described substratum, each component is formulated in water; And/or
The pH value 6 ± 0.5 of described substratum.
4. the purposes of the arbitrary described substratum of claim 1-3, for cultivate ocean, deep-sea thermophilic bacterium (
geobacillus sp.) 4j, produce amylase.
5. one kind prepare arbitrary described ocean, the deep-sea thermophilic bacterium of claim 1-3 (
geobacillus sp.) method of substratum of 4j, it is characterized in that, described method comprises:
A described Zulkovsky starch is carried out gelatinization by (), cooling;
B () is by described yeast powder, SODIUMNITRATE, sodium-chlor, anhydrous magnesium sulfate, magnesium chloride, Repone K, calcium chloride, dipotassium hydrogen phosphate, ferrous ammonium sulphate mixing;
C the solution that above-mentioned two steps obtain mixes by (), add nickel chloride solution and trace element;
Wherein, the consumption of each component arbitrary according to claim 1-3 described in.
6. one kind utilize ocean, deep-sea thermophilic bacterium (
geobacillus sp.) 4j produces diastatic method, it is characterized in that, described method comprises: utilize arbitrary described ocean, the culture medium culturing deep-sea thermophilic bacterium of claim 1-3 (
geobacillus sp.) 4j, thus produce amylase.
7. method as claimed in claim 6, is characterized in that, by ocean, deep-sea thermophilic bacterium (
geobacillus sp.) inoculum size of 4j 1-5% by volume inoculates into the arbitrary described substratum of claim 1-3,60 DEG C, static condition bottom fermentation cultivates 72-96h, carry out an oscillation treatment every 12h in quiescent culture process, will cultivate bacterium liquid and mix; After cultivating 72h and 96h, centrifugation fermented supernatant fluid, wherein containing amylase.
8. one kind for cultivate ocean, deep-sea thermophilic bacterium (
geobacillus sp.) test kit of 4j, it is characterized in that, described test kit comprises: the arbitrary described substratum of claim 1-3.
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