CN105647827A - Bacterial strain with high yield of enzyme for producing maltotriose, and screening and culturing method thereof - Google Patents
Bacterial strain with high yield of enzyme for producing maltotriose, and screening and culturing method thereof Download PDFInfo
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Abstract
The invention discloses a bacterial strain with a high yield of enzyme for producing maltotriose, and a screening and culturing method thereof. The bacterial strain is a mutant strain Metp-57 of Microbacterium imperiale, and the mutant strain is preserved in China General Microbiological Culture Collection Center, CGMCC, and the preservation number is CGMCC No.11536. The mutant strain Metp-57 is obtained through a plasma mutation method, and the genetic stability of Metp-57 is higher, compared with that of strains obtained through a conventional mutation method. The enzymatic activity of enzyme for producing maltotriose is higher. The output is greatly improved, compared with that of primary strain Mebl-012; and the production cost is reduced at the same time.
Description
Technical field
The present invention relates to the technical field of fermentable, it is that a kind of trisaccharide maltose forms the superior strain of enzyme and screening thereof and cultural method specifically.
Background technology
It is a kind of diastase with formation trisaccharide maltose ability that trisaccharide maltose forms enzyme, it is possible to from the ��-1,4-glycosidic link of inner hydrolyzed starch, thus produce trisaccharide maltose.
Trisaccharide maltose is a kind of Fructus Hordei Germinatus oligose. Trisaccharide maltose sweet taste is soft, and sugariness is low, viscosity height, infiltration is forced down, absorption easy to digest, anti-crystallization can be good, can obviously reduce freezing point of solution decline, promote that human body is to the absorption of calcium, can effectively suppress the growth of harmful bacteria in human intestinal, promote the breeding of human body probiotics, also can prevent protein denaturation, suppress age of starch, it is a kind of desirable healthy food material. Trisaccharide maltose can go directly enteron aisle, by the glycoside hydrolysis enzymic hydrolysis on mucous membrane of small intestine, rapid supplementing energy, can be used as the best endurance type functional beverage of sportsmen, heavy worker and long distance travelers. Trisaccharide maltose is also adapted in local flavor milky-drinks to apply, add the trisaccharide maltose of 6%��8%, can improve the nutrition and health care function of milky-drinks to a great extent in milky-drinks. Trisaccharide maltose has remarkable water absorbability and performance of keeping humidity, adds the trisaccharide maltose of less than 5%, can improve bread color and luster, make bread, homogeneous microstructure in bread as basic food, and performance of keeping humidity is good, and can slow down bread staling, extends shelf-lives and reaches 2��3d. The low sugariness of trisaccharide maltose, preferably heat-resisting, acid resistance, can be widely used in candy, frozen product, dessert, cake, chewing gum, cake, butter, cream, stirs cream, sweet beans filling etc.
The mode of production of current trisaccharide maltose is divided into two classes, and one is raw material taking pulullan polysaccharide, utilizes Pullulanase or isoamylase to carry out decomposing and obtaining, and the trisaccharide maltose purity height obtained by this method is the Perfected process preparing reagent and standard specimen. But Pu Lulan is expensive, be not thus suitable for the preparation of food raw material. Another kind is mainly obtained by trisaccharide maltose type amylase starch-splitting. First starch bacterial��-amylase is carried out post liquefaction, then form enzymic hydrolysis with trisaccharide maltose, it may also be useful to this hydrolysis process can obtain the syrup of higher trisaccharide maltose content.
It is prepare in trisaccharide maltose process the enzyme that can not obtain that trisaccharide maltose forms enzyme, and current trisaccharide maltose forms enzyme and still belongs to blank at home, and manufacturer does not produce, and technology is by external business monopoly.And the suitability for industrialized production that trisaccharide maltose forms enzyme is mainly produced by microbial fermentation processes, but the fermentation level of this organized enzyme is also quite low at present.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of trisaccharide maltose and forms the superior strain of enzyme and screening thereof and cultural method.
The present invention is the technical scheme that the technical problem existed in solution known technology is taked:
The trisaccharide maltose of the present invention forms the superior strain of enzyme, for the mutagenic strain Metp-57 of moth microbacterium Microbacteriumimperiale, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 28th, 2015, deposit number CGMCCNo.11536, preservation address: No. 1, Chaoyang District Beijing North Star West Road No. 3 Institute of Microorganism, Academia Sinica of institute.
The present invention can also adopt following technical measures:
The trisaccharide maltose of the present invention forms the screening method of the superior strain of enzyme, comprises the following steps:
(1) the product trisaccharide maltose that growth selection is good generates the moth microbacterium Mebl-012 inclined-plane bacterial strain of enzyme, makes 108The bacteria suspension of individual/mL;
(2) by the bacteria suspension in (1), draw 10 �� L on round iron plate, be placed in take helium as working gas, power 110W, in the atmospheric pressure at room plasma body mutagenesis system of working gas flow 10L/min, process distance is 2mm, processes 0s, 10s, 20s, 30s, 40s, 50s, 60s, 70s, 80s, 90s respectively, the spore suspension of process carries out gradient dilution to be coated with dull and stereotyped, makes lethality rate curve;
(3) according to the lethality rate curve of (2), select the irradiation time of high, medium and low three different lethal doses, the bacteria suspension of bacterial strain Mebl-012 is carried out plasma body mutagenesis;
(4) bacteria suspension of three different treatment times in (3) is mixed in the test tube that physiological saline is housed, mixes;
(5), by the mutagenesis spore suspension in (4), after carrying out gradient dilution, coat on the screening flat board containing starch respectively;
(6) by each resistant panel in (5) grows single bacterium colony switching inclined-plane after, with the inoculum size of 1%, it is inoculated in seed culture medium, 30 DEG C, 150r/min, after cultivating 45-50h, is inoculated in fermention medium with the inoculum size of 10% by seed liquor, 30 DEG C, 150r/min cultivates 3-5d;
(7) fermented liquid in (6) is got in centrifuge tube, the centrifugal 10min of 7000r/min, getting supernatant liquor is crude enzyme liquid, getting 1ml crude enzyme liquid joins in 2% Zulkovsky starch solution, 55 DEG C of water-baths 20 minutes, after crossing organic filter membrane, utilize the output of high effective liquid chromatography for measuring trisaccharide maltose, filter out, by this method, the superior strain Metp-57 that trisaccharide maltose forms enzyme.
Moth microbacterium Mebl-012 purchased from China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCCNo.1.1910.
In seed culture medium: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, all the other are distilled water, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min; In fermention medium: Zulkovsky starch 2%, bran powder 1%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, SODIUMNITRATE 0.5%, polysorbate40 0.1%, all the other are distilled water, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min.
The trisaccharide maltose of the present invention forms the cultural method of the superior strain of enzyme, comprises the following steps:
(1) actication of culture: moth microbacterium Metp-57 is forwarded to preservation isolation medium, 30 DEG C of constant temperature culture 3-7d;
(2) seed culture: the inclined-plane choosing well-grown, preparation concentration about 108The bacteria suspension of individual/mL, is inoculated in the 500mL triangular flask that 100mL seed culture medium is housed with the inoculum size of 1%, 30 DEG C, 150r/min, and 45-50h is cultivated in concussion;
(3) fermentation culture: by the seed culture fluid in (2), is inoculated in the 500mL triangular flask that 100mL fermention medium is housed with the inoculum size of 10%, 30 DEG C, 150r/min, and 3-5d is cultivated in concussion.
In preservation isolation medium: Zulkovsky starch 0.2%, peptone 1%, extractum carnis 0.5%, sodium-chlor 0.5%, foot portions distilled water is not supplied, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min; In seed culture medium: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, all the other are distilled water, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min; In fermention medium: Zulkovsky starch 2%, bran powder 1%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, SODIUMNITRATE 0.5%, polysorbate40 0.1%, all the other are distilled water, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min.
The advantage that the present invention has and positively effect be:
In the superior strain that the trisaccharide maltose of the present invention forms enzyme and screening, cultural method, mutagenic strain Metp-57 is obtained by plasma body mutagenesis method, higher than the bacterial strain genetic stability that conventional mutafacient system obtains, the enzyme that trisaccharide maltose forms enzyme is lived higher, its output improves a lot relative to the output of starting strain Mebl-012, also reduces production cost simultaneously.
Embodiment
By the following examples the technical scheme of the present invention is described in detail.
The trisaccharide maltose of the present invention forms the superior strain of enzyme, for the mutagenic strain Metp-57 of moth microbacterium Microbacteriumimperiale, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCCNo.11536.
The product trisaccharide maltose that filters out from soil forms the bacterial strain Mebl-012 of enzyme, moth microbacterium Mebl-012 purchased from China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCCNo.1.1910. .
Mebl-012 thalli morphology: thalline is Gram-negative bacteria, and somatic cells is shaft-like. Bacterium colony be orange-yellow, protuberance, the smooth of the edge neat, have viscosity.
Mebl-012 cultural characteristic: this bacterium optimum growth temperature is 30 DEG C, the most suitable growth pH is 7.0-7.2; Being 140-150r/min in shaking speed, when cultivating 2-3d, it is the highest that trisaccharide maltose forms production of enzyme, and in fermenting process, the generation that trisaccharide maltose forms enzyme is had remarkably influenced by temperature.
The characteristic of mutagenic strain Metp-57:
Thalli morphology: thalline is Gram-negative bacteria, and somatic cells is shaft-like. Bacterium colony be orange-yellow, protuberance, the smooth of the edge neat, have viscosity.
Cultural characteristic: this bacterium optimum growth temperature is 30 DEG C, the most suitable growth pH is 7.1; Being 150r/min in shaking speed, when cultivating 3d, trisaccharide maltose forms enzyme enzyme and lives the highest.
Can grow in containing the substratum of starch, drip to flat board and add iodine liquid and can form transparent circle in periphery of bacterial colonies.
Embodiment 1:
Taking moth microbacterium Mebl-012 as starting strain, adopt atmospheric pressure at room plasma body mutagenesis technology screening bacterial strain Metp-57, comprise the following steps:
(1) the product trisaccharide maltose that growth selection is good forms the moth microbacterium Mebl-012 inclined-plane bacterial strain of enzyme, inoculates in a ring to seed liquor and makes 108The bacteria suspension of individual/mL;
(2) by the spore suspension in (1), drawing 10 �� L in diameter with shifting liquid rifle is on the round iron plate of 1cm, it is placed in using helium as working gas, power is 110W, in the atmospheric pressure at room plasma body mutagenesis system of working gas flow 10L/min, process distance is 2mm, processes 0s, 10s, 20s, 30s, 40s, 50s, 60s, 70s, 80s, 90s respectively, the bacteria suspension of process carries out gradient dilution to be coated with dull and stereotyped, makes lethality rate curve;
(3) according to the lethality rate curve of (2), select the irradiation time of 30s, 50s, 70s tri-different lethal doses, the bacteria suspension of bacterial strain Mebl-012 is carried out plasma body mutagenesis;
(4) bacteria suspension of three different treatment times in (3) is mixed in the test tube that physiological saline is housed, mixes;
(5) by the mutagenic bacteria suspension in (4), gradient dilution 10 is carried out-1-10-6, get 10-4��10-5��10-6The bacteria suspension of three extent of dilution, coats containing on starch coloration flat board respectively;
(6) by behind single bacterium colony switching inclined-plane big for each dull and stereotyped upper starch transparent circle in (5), with the inoculum size of 1%, it is inoculated in seed culture medium, 30 DEG C, 140r/min, after cultivating 50h, is inoculated in fermention medium with the inoculum size of 10% by seed liquor, 30 DEG C, 150r/min cultivates 3d; Seed culture medium consists of: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, and all the other are distilled water, pH7.0,121 DEG C of high pressure steam sterilization 20min; Described fermention medium consists of: Zulkovsky starch 2%, bran powder 1%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, SODIUMNITRATE 0.5%, polysorbate40 0.1%, all the other are distilled water, pH7.0,121 DEG C of high pressure steam sterilization 20min.
(7) fermented liquid in (6) is got in centrifuge tube, the centrifugal 10min of 7000r/min, get centrifuged supernatant 1ml, add in the large size test tube that 10ml2% Zulkovsky starch is housed, water-bath 20min on the water-bath shaking table of 50 DEG C, get after enzymolysis solution crosses organic filter membrane, utilize the output of high effective liquid chromatography for measuring trisaccharide maltose, namely filter out trisaccharide maltose by this method and form enzyme enzyme and live high mutagenic strain Metp-57.
The trisaccharide maltose of the present invention forms the cultural method of the superior strain of enzyme, comprises the following steps:
1, the preparation of substratum
(1) preservation isolation medium: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, all the other are distilled water, pH7.0,121 DEG C of high pressure steam sterilization 20min;
(2) seed culture medium: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, all the other are distilled water, pH7.0,121 DEG C of high pressure steam sterilization 20min;
(3) fermention medium: Zulkovsky starch 2%, bran powder 1%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, SODIUMNITRATE 0.5%, polysorbate40 0.1%, all the other are distilled water, pH7.0,121 DEG C of high pressure steam sterilization 20min.
2, actication of culture
The bacterial classification Metp-57 that glycerine preserves is forwarded to preservation isolation medium inclined-plane, in constant incubator, cultivates 15d for 30 DEG C;
3, seed culture
The activated inclined plane that growth selection is good, is inoculated in the seed culture medium that to be equipped with in above-mentioned 1 obtained, the in-built 100mL substratum of 500mL triangular flask, and in 30 DEG C, 150r/min, cultivates 50h;
4, fermentation culture
Seed liquor obtained in above-mentioned steps 3 is inoculated in fermention medium with 10% (v/v) inoculum size, the in-built 80mL substratum of 500mL triangular flask, in 30 DEG C, fermentation culture 5d when 150r/min.
Detected result: trisaccharide maltose generates the output of enzyme to moth microbacterium MicrobacteriumimperialeMetp-57 on fermention medium is 241.32U/mL, than the output 116.43U/mL of starting strain Mebl-012, it is to increase 107.26%.
Embodiment 2:
Taking moth microbacterium Mebl-012 as starting strain, adopt atmospheric pressure at room plasma body mutagenesis technology screening bacterial strain Metp-57, comprise the following steps:
(1) the product trisaccharide maltose that growth selection is good forms the moth microbacterium Mebl-012 inclined-plane bacterial strain of enzyme, inoculates in a ring to seed liquor and makes 108The bacteria suspension of individual/mL;
(2) by the spore suspension in (1), drawing 10 �� L in diameter with shifting liquid rifle is on the round iron plate of 1cm, it is placed in using helium as working gas, power is 110W, in the atmospheric pressure at room plasma body mutagenesis system of working gas flow 10L/min, process distance is 2mm, processes 0s, 10s, 20s, 30s, 40s, 50s, 60s, 70s, 80s, 90s respectively, the bacteria suspension of process carries out gradient dilution to be coated with dull and stereotyped, makes lethality rate curve;
(3) according to the lethality rate curve of (2), select the irradiation time of 10s, 40s, 80s tri-different lethal doses, the bacteria suspension of bacterial strain Mebl-012 is carried out plasma body mutagenesis;
(4) bacteria suspension of three different treatment times in (3) is mixed in the test tube that physiological saline is housed, mixes;
(5) by the mutagenic bacteria suspension in (4), gradient dilution 10 is carried out-1-10-6, get 10-4��10-5��10-6The bacteria suspension of three extent of dilution, coats containing on starch coloration flat board respectively;
(6) by behind single bacterium colony switching inclined-plane big for each dull and stereotyped upper starch transparent circle in (5), with the inoculum size of 1%, it is inoculated in seed culture medium, 30 DEG C, 150r/min, after cultivating 45h, is inoculated in fermention medium with the inoculum size of 10% by seed liquor, 30 DEG C, 150r/min cultivates 2d; Seed culture medium consists of: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, and all the other are distilled water, pH7.2,121 DEG C of high pressure steam sterilization 20min; Described fermention medium consists of: Zulkovsky starch 2%, bran powder 1%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, SODIUMNITRATE 0.5%, polysorbate40 0.1%, all the other are distilled water, pH7.2,121 DEG C of high pressure steam sterilization 20min.
(7) fermented liquid in (6) is got in centrifuge tube, the centrifugal 10min of 7000r/min, get centrifuged supernatant 1ml, add in the large size test tube that 10ml2% Zulkovsky starch is housed, water-bath 20min on the water-bath shaking table of 50 DEG C, get after enzymolysis solution crosses organic filter membrane, utilize the output of high effective liquid chromatography for measuring trisaccharide maltose, namely filter out trisaccharide maltose by this method and form enzyme enzyme and live high mutagenic strain Metp-57.
The trisaccharide maltose of the present invention forms the cultural method of the superior strain of enzyme, comprises the following steps:
1, the preparation of substratum
(1) preservation isolation medium: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, all the other are distilled water, pH7.2,121 DEG C of high pressure steam sterilization 20min;
(2) seed culture medium: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, all the other are distilled water, pH7.2,121 DEG C of high pressure steam sterilization 20min;
(3) fermention medium: Zulkovsky starch 2%, bran powder 1%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, SODIUMNITRATE 0.5%, polysorbate40 0.1%, all the other are distilled water, pH7.2,121 DEG C of high pressure steam sterilization 20min.
2, actication of culture
The bacterial classification Metp-57 that glycerine preserves is forwarded to preservation isolation medium inclined-plane, in constant incubator, cultivates 7d for 30 DEG C;
3, seed culture
The activated inclined plane that growth selection is good, is inoculated in the seed culture medium that to be equipped with in above-mentioned 1 obtained, the in-built 100mL substratum of 500mL triangular flask, and in 30 DEG C, 150r/min, cultivates 45h;
4, fermentation culture
Seed liquor obtained in above-mentioned steps 3 is inoculated in fermention medium with 10% (v/v) inoculum size, the in-built 80mL substratum of 500mL triangular flask, in 30 DEG C, fermentation culture 3d when 150r/min.
Detected result: trisaccharide maltose generates the output of enzyme to moth microbacterium MicrobacteriumimperialeMetp-57 on fermention medium is 238.49U/mL, than the output 116.43U/mL of starting strain Mebl-012, it is to increase 104.84%.
Embodiment 3:
Taking moth microbacterium Mebl-012 as starting strain, adopt atmospheric pressure at room plasma body mutagenesis technology screening bacterial strain Metp-57, comprise the following steps:
(1) the product trisaccharide maltose that growth selection is good forms the moth microbacterium Mebl-012 inclined-plane bacterial strain of enzyme, inoculates in a ring to seed liquor and makes 108The bacteria suspension of individual/mL;
(2) by the spore suspension in (1), drawing 10 �� L in diameter with shifting liquid rifle is on the round iron plate of 1cm, it is placed in using helium as working gas, power is 110W, in the atmospheric pressure at room plasma body mutagenesis system of working gas flow 10L/min, process distance is 2mm, processes 0s, 10s, 20s, 30s, 40s, 50s, 60s, 70s, 80s, 90s respectively, the bacteria suspension of process carries out gradient dilution to be coated with dull and stereotyped, makes lethality rate curve;
(3) according to the lethality rate curve of (2), select the irradiation time of 20s, 60s, 80s tri-different lethal doses, the bacteria suspension of bacterial strain Mebl-012 is carried out plasma body mutagenesis;
(4) bacteria suspension of three different treatment times in (3) is mixed in the test tube that physiological saline is housed, mixes;
(5) by the mutagenic bacteria suspension in (4), gradient dilution 10 is carried out-1-10-6, get 10-4��10-5��10-6The bacteria suspension of three extent of dilution, coats containing on starch coloration flat board respectively;
(6) by behind single bacterium colony switching inclined-plane big for each dull and stereotyped upper starch transparent circle in (5), with the inoculum size of 1%, it is inoculated in seed culture medium, 30 DEG C, 150r/min, after cultivating 48h, is inoculated in fermention medium with the inoculum size of 10% by seed liquor, 30 DEG C, 150r/min cultivates 2.5d; Seed culture medium consists of: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, and all the other are distilled water, pH7.1,121 DEG C of high pressure steam sterilization 20min; Described fermention medium consists of: Zulkovsky starch 2%, bran powder 1%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, SODIUMNITRATE 0.5%, polysorbate40 0.1%, all the other are distilled water, pH7.1,121 DEG C of high pressure steam sterilization 20min.
(7) fermented liquid in (6) is got in centrifuge tube, the centrifugal 10min of 7000r/min, get centrifuged supernatant 1ml, add in the large size test tube that 10ml2% Zulkovsky starch is housed, water-bath 20min on the water-bath shaking table of 50 DEG C, get after enzymolysis solution crosses organic filter membrane, utilize the output of high effective liquid chromatography for measuring trisaccharide maltose, namely filter out trisaccharide maltose by this method and form enzyme enzyme and live high mutagenic strain Metp-57.
The trisaccharide maltose of the present invention forms the cultural method of the superior strain of enzyme, comprises the following steps:
1, the preparation of substratum
(1) preservation isolation medium: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, all the other are distilled water, pH7.1,121 DEG C of high pressure steam sterilization 20min;
(2) seed culture medium: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, all the other are distilled water, pH7.1,121 DEG C of high pressure steam sterilization 20min;
(3) fermention medium: Zulkovsky starch 2%, bran powder 1%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, SODIUMNITRATE 0.5%, polysorbate40 0.1%, all the other are distilled water, pH7.1,121 DEG C of high pressure steam sterilization 20min.
2, actication of culture
The bacterial classification Metp-57 that glycerine preserves is forwarded to preservation isolation medium inclined-plane, in constant incubator, cultivates 10d for 30 DEG C;
3, seed culture
The activated inclined plane that growth selection is good, is inoculated in the seed culture medium that to be equipped with in above-mentioned 1 obtained, the in-built 100mL substratum of 500mL triangular flask, and in 30 DEG C, 150r/min, cultivates 48h;
4, fermentation culture
Seed liquor obtained in above-mentioned steps 3 is inoculated in fermention medium with 10% (v/v) inoculum size, the in-built 80mL substratum of 500mL triangular flask, in 30 DEG C, fermentation culture 4d when 150r/min.
Detected result: trisaccharide maltose generates the output of enzyme to moth microbacterium MicrobacteriumimperialeMetp-57 on fermention medium is 243.92U/mL, than the output 116.43U/mL of starting strain Mebl-012, it is to increase 109.5%.
The above, it it is only the better embodiment of the present invention, not the present invention is done any restriction in form, although the present invention is with better embodiment openly as above, but, and be not used to limit the present invention, any those skilled in the art, do not departing within the scope of technical solution of the present invention, certainly the technology contents of announcement can be utilized to make a little change or modification, become the equivalent embodiment of equivalent variations, in every case it is the content not departing from technical solution of the present invention, the any simple modification above embodiment done according to the technical spirit of the present invention, equivalent variations and modification, all belong in the scope of technical solution of the present invention.
Claims (6)
1. the superior strain of a trisaccharide maltose formation enzyme, for the mutagenic strain Metp-57 of moth microbacterium Microbacteriumimperiale, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCCNo.11536.
2. trisaccharide maltose forms a screening method for the superior strain of enzyme, comprises the following steps:
(1) the product trisaccharide maltose that growth selection is good generates the moth microbacterium Mebl-012 inclined-plane bacterial strain of enzyme, makes 108The bacteria suspension of individual/mL;
(2) by the bacteria suspension in (1), draw 10 �� L on round iron plate, be placed in take helium as working gas, power 110W, in the atmospheric pressure at room plasma body mutagenesis system of working gas flow 10L/min, process distance is 2mm, processes 0s, 10s, 20s, 30s, 40s, 50s, 60s, 70s, 80s, 90s respectively, spore suspension after process carries out gradient dilution to be coated with dull and stereotyped, makes lethality rate curve;
(3) according to the lethality rate curve of (2), select the irradiation time of high, medium and low three different lethal doses, the bacteria suspension of bacterial strain Mebl-012 is carried out plasma body mutagenesis;
(4) bacteria suspension of three different treatment times in (3) is mixed in the test tube that physiological saline is housed, mixes;
(5), by the mutagenesis spore suspension in (4), after carrying out gradient dilution, coat on the screening flat board containing starch respectively;
(6) the single bacterium colony grown in each resistant panel in (5) is transferred behind inclined-plane, with the inoculum size of 1%, it is inoculated in seed culture medium, 30 DEG C, 150r/min, after cultivating 45-50h, with the inoculum size of 10%, seed liquor being inoculated in fermention medium, 30 DEG C, 150r/min cultivates 3-5d;
(7) fermented liquid in (6) is got in centrifuge tube, the centrifugal 10min of 7000r/min, getting supernatant liquor is crude enzyme liquid, getting 1ml crude enzyme liquid joins in 2% Zulkovsky starch solution, 55 DEG C of water-baths 20 minutes, after crossing organic filter membrane, utilize the output of high effective liquid chromatography for measuring trisaccharide maltose, filter out, by this method, the superior strain Metp-57 that trisaccharide maltose forms enzyme.
3. trisaccharide maltose according to claim 2 forms the superior strain of enzyme, it is characterised in that: moth microbacterium Mebl-012 purchased from China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterium numbering CGMCCNo.1.1910.
4. trisaccharide maltose according to claim 3 forms the screening method of the superior strain of enzyme, it is characterised in that: in seed culture medium: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, all the other are distilled water, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min; In fermention medium: Zulkovsky starch 2%, bran powder 1%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, SODIUMNITRATE 0.5%, polysorbate40 0.1%, all the other are distilled water, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min.
5. trisaccharide maltose forms a cultural method for the superior strain of enzyme, comprises the following steps:
(1) actication of culture: moth microbacterium Metp-57 is forwarded to preservation isolation medium, 30 DEG C of constant temperature culture 3-7d;
(2) seed culture: the inclined-plane choosing well-grown, preparation concentration about 108The bacteria suspension of individual/mL, is inoculated in the 500mL triangular flask that 100mL seed culture medium is housed with the inoculum size of 1%, 30 DEG C, 150r/min, and 45-50h is cultivated in concussion;
(3) fermentation culture: by the seed culture fluid in (2), is inoculated in the 500mL triangular flask that 100mL fermention medium is housed with the inoculum size of volume ratio 10%, 30 DEG C, 150r/min, and 3-5d is cultivated in concussion.
6. trisaccharide maltose according to claim 5 forms the cultural method of the superior strain of enzyme, it is characterised in that: in preservation isolation medium: Zulkovsky starch 0.2%, peptone 1%, extractum carnis 0.5%, sodium-chlor 0.5%, all the other are distilled water, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min; In seed culture medium: extractum carnis 0.5%, peptone 1%, sodium-chlor 0.5%, foot portions distilled water is not supplied, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min; In fermention medium: Zulkovsky starch 2%, bran powder 1%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium primary phosphate 0.5%, magnesium sulfate 0.2%, SODIUMNITRATE 0.5%, polysorbate40 0.1%, all the other are distilled water, pH7.0-7.2,121 DEG C of high pressure steam sterilization 20min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020177456A1 (en) * | 2019-03-05 | 2020-09-10 | 山东安华生物医药股份有限公司 | Mutagenic strain of lactobacillus plantarum, mutagenesis method therefor and application thereof |
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| CN102510900A (en) * | 2009-07-01 | 2012-06-20 | 天野酶株式会社 | Maltotriocil transferase, process for production thereof, and use thereof |
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| CN102510900A (en) * | 2009-07-01 | 2012-06-20 | 天野酶株式会社 | Maltotriocil transferase, process for production thereof, and use thereof |
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| M. CANTARELLA等: "Nicotinic acid bio-production by Microbacterium imperiale CBS 489-74: Effect of 3-cyanopyridine and temperature on amidase activity", 《PROCESS BIOCHEMISTRY》 * |
| Y TAKASAKI等: "Maltotriose-producing Amylase from Microbacterium imperial", 《JOURNAL OF THE AGRICULTURAL CHEMICAL SOCIETY OF JAPAN》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020177456A1 (en) * | 2019-03-05 | 2020-09-10 | 山东安华生物医药股份有限公司 | Mutagenic strain of lactobacillus plantarum, mutagenesis method therefor and application thereof |
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