CN1970732A - Active strain of high-activity saccharifying enzyme, enzyme preparation, and their preparation method and use - Google Patents
Active strain of high-activity saccharifying enzyme, enzyme preparation, and their preparation method and use Download PDFInfo
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- CN1970732A CN1970732A CN 200610102056 CN200610102056A CN1970732A CN 1970732 A CN1970732 A CN 1970732A CN 200610102056 CN200610102056 CN 200610102056 CN 200610102056 A CN200610102056 A CN 200610102056A CN 1970732 A CN1970732 A CN 1970732A
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 76
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 75
- 238000002360 preparation method Methods 0.000 title claims description 34
- 230000000694 effects Effects 0.000 title abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 47
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 13
- 239000007787 solid Substances 0.000 claims abstract description 6
- 230000001580 bacterial effect Effects 0.000 claims description 50
- 235000015099 wheat brans Nutrition 0.000 claims description 44
- 238000000855 fermentation Methods 0.000 claims description 24
- 230000004151 fermentation Effects 0.000 claims description 24
- 238000004519 manufacturing process Methods 0.000 claims description 22
- 239000001963 growth medium Substances 0.000 claims description 14
- 239000012669 liquid formulation Substances 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 11
- 238000009423 ventilation Methods 0.000 claims description 11
- 235000021419 vinegar Nutrition 0.000 claims description 10
- 239000000052 vinegar Substances 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 230000004913 activation Effects 0.000 claims description 8
- 235000014101 wine Nutrition 0.000 claims description 8
- 238000011218 seed culture Methods 0.000 claims description 6
- 150000007524 organic acids Chemical class 0.000 claims description 5
- 238000007670 refining Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 2
- 239000003242 anti bacterial agent Substances 0.000 claims 1
- 229940088710 antibiotic agent Drugs 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 9
- 229920002472 Starch Polymers 0.000 abstract description 6
- 235000019698 starch Nutrition 0.000 abstract description 6
- 239000008107 starch Substances 0.000 abstract description 6
- 239000003795 chemical substances by application Substances 0.000 abstract description 5
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 23
- 239000000843 powder Substances 0.000 description 20
- 239000002054 inoculum Substances 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 244000061456 Solanum tuberosum Species 0.000 description 10
- 235000002595 Solanum tuberosum Nutrition 0.000 description 10
- 102100022624 Glucoamylase Human genes 0.000 description 8
- 239000000047 product Substances 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 102000035195 Peptidases Human genes 0.000 description 6
- 108091005804 Peptidases Proteins 0.000 description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 6
- 235000011130 ammonium sulphate Nutrition 0.000 description 6
- 235000019833 protease Nutrition 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 238000004659 sterilization and disinfection Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 239000000049 pigment Substances 0.000 description 5
- 229910019142 PO4 Inorganic materials 0.000 description 4
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 229910052700 potassium Inorganic materials 0.000 description 4
- 239000011591 potassium Substances 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 210000000582 semen Anatomy 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000010563 solid-state fermentation Methods 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 244000046052 Phaseolus vulgaris Species 0.000 description 2
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- -1 coefficient is 70% Substances 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000006052 feed supplement Substances 0.000 description 2
- 238000012262 fermentative production Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
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- 229920001817 Agar Polymers 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
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- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 description 1
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- 244000046109 Sorghum vulgare var. nervosum Species 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a saccharifying enzyme agent and making method and application with reserving number at CGMCC No.1818 for high-saccharifying enzyme aspergillus niger mutant IN7-31, which is characterized by the following: improving starch utilizing rate effectively; establishing new solid and liquid ferment method to produce saccharifying enzyme agent with enzyme activity at 7000-12000 unit/g (mg/h) as solid saccharifying enzyme agent and 2200-4200 unit/ml as liquid saccharifying enzyme agent.
Description
Technical field
The present invention relates to enzyme, belong to saccharifying enzyme, specifically is a kind of high saccharifying enzyme bacterial classification alive, zymin and its production and application.
Technical background
Enzyme engineering is a bionic important component part, and saccharifying enzyme is one of emphasis direction of enzyme engineering research at all times.At present, the theoretical investigation of saccharifying enzyme mainly concentrates on the aspects such as polytypism, catalytic reaction kinetics, enzyme gene and metabolic regulation thereof of enzymatic structure, and applied research still concentrates on the seed selection of saccharifying enzyme high dynamic strain, the immobilization of enzyme and the aspects such as the technology of applying of zymin.
Aspect seed selection of saccharifying enzyme high dynamic strain and zymin utilisation technology, in China a research climax appearred once, and obtain a collection of achievement, and had some saccharifying enzyme high dynamic strains to realize industrialization production, effectively promoted the development of related industries.Yet for various reasons, the research work of this respect is in a low tide one over nearly 10 years, and relevant achievement report falls sharply, so caused current research work to lag behind the difficult situation of production development demand.At present, saccharifying enzyme research with the basic situation of using is:
1, enzyme activity unit disunity, the GB/T1803-93 of China National Light Industrial Products Department standard (China National Light Industrial Products Department's ministerial standard: industrial saccharifying amylase quality standard) regulation: " under optimum condition, per hour converted starch matter substrate generation 1mg glucose is called 1 enzyme unit alive " (definition 1).But the people is also arranged with " under optimum condition, per minute converted starch matter substrate produces 1ug glucose and is defined as 1 enzyme unit alive " (definition 2).Two enzymes unit difference of living are bigger, caused the chaos of conception and used mistake, some bibliographical information even self-contradictory result occurs.
2, owing to strain improvement process length consuming time, workload is big, and directional property is poor, and success ratio is low, and manpower, financial resources, the energy to this research drops into fewer and feweri in recent years.Therefore, have only a few studies report over nearly 10 years, for example 1999, the saccharifying enzyme superior strain fermented liquid glucoamylase enzyme work of its seed selections of report such as the paddy Hai Xian of the light institute in Wuxi was 1736 units/ml, amounted to definition 2 and was expressed as 28991 units/ml.
3, present, the mode of production of domestic Glucoamylase preperation mainly contains two kinds, i.e. liquid fermentation method and solid state fermentation.The saccharifying enzymic activity of liquid fermentation method is in following level: for example, Dalian distillery in 1998 reports that its fermentative production level is 890 units/ml, amounts to definition 2 and is expressed as 14863 units/ml.And for example, biotechnology company limited of calendar year 2001 Jiangxi Hao De group moral saccharifying enzyme production level is on average at 1800 units/ml, amount to definition 2 and be expressed as 30060 units/ml, can be increased to 2280～2400 units/ml by the fed-batch fermentation mode, amount to definition 2 and be expressed as 38076～40080 units/ml.Saccharifying enzymic activity is generally not high.Adopt solid state fermentation, saccharifying enzyme leavened prod to comprise two kinds in wheat bran and Daqu, wheat bran is the solid-state Glucoamylase preperation by the higher bacterial strain preparation of saccharifying enzymic activity, the solid-state mixed culture fermentation agent that Daqu then is made up of multiple functional microorganism.The saccharifying enzyme of wheat bran is lived many at 2400 units/g, amounts to definition 2 and is expressed as that 40080 units/below the g, the saccharifying enzyme of Daqu is lived then lower, generally at 1000 units/g, amounts to definition 2 and is expressed as about 16700 units/g.For example, the wheat bran dominant bacteria saccharifying enzymic activity of western Hunan He Xi aromatic vinegar factory use in 2006 is 1798 units/g, amounts to definition 2 and is expressed as 30026 units/g.The saccharifying enzyme work of Fenyang wine Daqu on average about 1200 units/g, is amounted to definition 2 and is expressed as 20040 units/g.In addition, most proteinase of moldy bran are produced bacterial strains can produce a large amount of pigments, and Cheng Quhou has unpleasant mouldy and corrupe, is having a strong impact on the application of Glucoamylase preperation.
4, present, Glucoamylase preperation is widely used in the production of products such as alcohol, liquor, yellow rice wine, microbiotic, organic acid, vinegar.Refining saccharifying enzyme not only applies to also be used by some food-processing industries in a lot of fermentative production, for example has to be reported in to use histological structure and the body state of saccharifying enzyme degraded starch raw material to improve product in the ice cream production.The then production processes that are used for the traditional fermentation product of crude zyme preparation (for example Daqu or wheat bran), for example the Fenyang wine in Shanxi, old vinegar etc. are brewageed product and just are to use special Daqu as its saccharifying ferment more.
But, no matter be the liquid state fermentation or the Glucoamylase preperation of solid state fermentation preparation, the subject matter of current existence is that the saccharifying enzymic activity of production bacterial strain is not high, especially when producing solid-state Glucoamylase preperation, can produce unpleasant mouldy and corrupe and a large amount of pigment, have a strong impact on the application of homomorphism Glucoamylase preperation.
Summary of the invention
The present invention aims to provide the bacterial classification that a kind of high saccharifying enzyme is lived, this bacterial classification is the saccharifying enzymic activity height not only, and has that pigment is few, the characteristics of no mouldy and corrupe: the live application of liquid formulation and preparation method thereof and two kinds of preparations of wheat bran and high saccharifying enzyme of living of high saccharifying enzyme is provided simultaneously.
The bacterial classification that a kind of high saccharifying enzyme provided by the invention is lived, be to select aspergillus niger As3.4309 for use, by plasma mutagenesis, lithium chloride～repeatedly cross processing of mutafacient system such as ultraviolet compounded mutagenesis, the high saccharifying enzyme live strain aspergillus niger IN7-31 of final acquisition, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on September 18th, 2006, and preserving number is CGMCC No.1818; Classification name: aspergillus niger (Aspergillus niger).
This bacterial strain has following feature:
It is very fast to grow on the potato substratum, and colony diameter can reach 5～6cm in 7 days, and colony colour is a yellow-white, culture medium flat plate back side non-pigment, no mouldy and corrupe.
The visible mycelia of observation is very short down for opticmicroscope, and spore is less, and mycelia has tabula, stigma bilayer, conidium sphere, 4～7 microns of diameters.
The wheat bran culture does not have mouldy and corrupe, and glucoamylase enzyme work is about 7000～12000 units/g and (amounts to into unit 2 and be expressed as 116900～200400 units/g), improved about 3.5 times than the work of starting strain glucoamylase enzyme.
This bacterial strain test tube 5 enzymatic productivities that go down to posterity do not have considerable change, and it is stable to produce the enzyme proterties.
A kind of high saccharifying enzyme provided by the invention wheat bran of living, adopting bran mass and preserving number is that the bacterial classification of CGMCC No.1818 is cultivated under appropriate condition and made.
The live preparation method of wheat bran of a kind of high saccharifying enzyme provided by the invention is characterized in that comprising the steps: that (1) is that the bacterial classification of CGMCC No.1818 is slant medium activation 2～4 times with preserving number; (2) the activatory bacterial classification is inserted the wheat bran seed culture medium, under 25～33 ℃ of temperature, cultivate and made solid state bacterial in 24～96 hours; (3) solid state bacterial is inserted in the bran mass, ventilating to cultivating under 25～33 ℃ of temperature promptly made high saccharifying enzyme wheat bran alive in 70～100 hours.
The slant medium that uses in the described step (1) can be potato substratum (being the PDA substratum); The wheat bran seed culture medium that uses in the step (2) can be to count by weight to comprise: 100 parts in wheat bran, 100～120 parts in water; The bran mass that step (3) is used can be to count to comprise 95～100 parts in wheat bran 0～5 part on rice husk, 3～5 parts of W-Gums, 0.5～1 part in ammonium sulfate, 0.3～0.5 part of potassium primary phosphate, 100～120 parts in water by weight.
Culture temperature is preferably 28～30 ℃ in described step (2), (3).
Inoculum size is 5～10% in the described step (3); Concrete optimum condition is cultivated in described ventilation: ventilation is 1: 0.3～1: 0.5, ventilates to cultivating after 36～54 hours, turns over Qu Yici, and regulating ventilation is 1: 0.5～1: 1, cultivates altogether promptly to make high enzyme wheat bran alive in 70～100 hours.
The purposes of a kind of high saccharifying enzyme of the present invention wheat bran alive, this wheat bran can be widely used in making wine, making in the production of products such as vinegar, alcohol, organic acid, because this wheat bran does not have mouldy and corrupe and chromogenesis not, especially be suitable for the brewageing in the production of various liquor, yellow rice wine and vinegar.
A kind of high saccharifying enzyme provided by the invention liquid formulation of living, it is characterized in that adopting liquid state fermentation substratum and preserving number is that the bacterial classification of CGMCC No.1818 is cultivated under appropriate condition and made.
The live preparation method of liquid formulation of a kind of high saccharifying enzyme provided by the invention is characterized in that comprising the steps: that (1) is that the bacterial classification of CGMCC No.1818 is slant medium activation 2～4 times with preserving number; (2) the activatory bacterial classification is inserted liquid bacterium culture medium, under 25～33 ℃ of temperature, cultivate and made liquid bacterial classification in 24～72 hours; (3) liquid bacterial classification is inserted in the liquid state fermentation substratum, control pH is 5～7, and under 25～33 ℃ of temperature, ventilation, stir culture promptly made high saccharifying enzyme liquid formulation alive in 50～100 hours.
The slant medium that uses in the described step (1) can be potato substratum (being the PDA substratum); The liquid bacterium culture medium that uses in the step (2) can be Cha Shi substratum or potato substratum; The liquid state fermentation substratum that uses in the step (3) can be to count by weight to comprise: 6～12 parts of Semen Maydis powder, 1～3 part of bean powder, 1～3 part in wheat bran, 0.5～3 part in ammonium sulfate, 0.5～1.5 part of dipotassium hydrogen phosphate, 0.5～1.5 part of potassium primary phosphate, 85～92 parts in water.
Described step (2) concrete operations are: bacterial classification potato slope go down to posterity the activation twice after, wash spore with sterilized water, be seeded in the liquid bacterium culture medium preferred culture condition: 28～30 ℃, 80～120rpm shaking table are cultivated and were promptly made liquid bacterial classification in 36～48 hours.
Described step (3) concrete operations are: by 5～10% inoculum size inoculation fermentation substratum, the control air flow is 1: 0.8～1: 1.2, and dissolved oxygen is 10%～20%.
Culture temperature is preferably 28～30 ℃ in described step (2), (3).
PH is preferably 6 in the described step (3).
The purposes of a kind of high saccharifying enzyme of the present invention liquid formulation alive, this kind liquid formulation is mainly used in the refining saccharifying enzyme of preparation, and the application in productions such as wine brewing, wine vinegar, alcohol, organic acid, microbiotic.
Advantage compared with prior art of the present invention and effect:
High saccharifying enzyme live strain IN7-31 growth velocity of the present invention and starting strain As3.4309 are more or less the same, but produce enzyme speed then apparently higher than As3.4309.
IN7-31 bacterial strain saccharifying enzymic activity height, can reach the bent powder of 7000～12000 units/g (amount to into unit 2 and be expressed as the bent powder of 116900～200400 units/g) by the proteinase of moldy bran enzyme work of this strain preparation, (amount to into unit 2 and be expressed as 36740～70140 units/ml) and can reach 2200～4200 units/ml by the glucoamylase fermentation broth glucoamylase enzyme work of this bacterial classification by the liquid submerged fermentation preparation.
The liquid submerged fermentation production technique of IN7-31 bacterial strain is easy, and production cost is low, and the fermented liquid glucoamylase enzyme is lived high, is easy to extract and purifying.
The wheat bran of IN7-31 bacterial strain preparation also has characteristics such as pigment is few, no mouldy and corrupe except that the saccharifying enzymic activity height, deposit 3 months alive only forfeitures about 30% of enzyme under shady and cool condition, is very suitable for brewageing producing and uses.
Description of drawings
The colonial morphology color comparison of Fig. 1 IN7-31 and As3.4309
Among the figure, the IN7-31 colony colour is a yellow-white, and the As3.4309 colony colour is a Vandyke brown.
Bacterial classification IN7-31 of the present invention is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on September 18th, 2006, and preserving number is CGMCC No.1818.
Embodiment
Embodiment 1 aspergillus niger IN7-31 compares with the bacterium aspergillus niger As3.4309 production traits of setting out
1. bacterial classification
Aspergillus niger IN7-31, aspergillus niger As3.4309
2. substratum
Slant medium: potato substratum (PDA), 121 ℃, 20min sterilization.
Wheat bran seed culture medium: wheat bran: water=1: 1,150mL triangular flask packing 10g substratum, 8 layers of gauze seal, 121 ℃, 90min sterilization.
Bran mass: wheat bran: water=1: 1,1000mL Porcelain Jar packing 200g substratum, cotton yarn cloth seals, 121 ℃, 120min sterilization.
3. the preparation of high glucoamylase enzyme wheat bran alive
The preparation of inoculum: inclined-plane twice Aspergillus niger strain IN7-31 and the As3.4309 of activation that go down to posterity is inoculated in the wheat bran seed culture medium respectively with certain inoculum size, cultivates for 28 ℃ and made the production bacterial classification in 72 hours.
The preparation of wheat bran: IN7-31 and As3.4309 are produced bacterial classification be inoculated in bran mass respectively with 5% inoculum size, promptly every cylinder inoculation 10g produces bacterial classification (As3.4309 organizes in contrast).Place 28 ℃ of culturing room, ventilate from the false bottom of cylinder and cultivate, ventilation is 1: 0.5, cultivates and turns over Qu Yici after 48 hours, and ventilation was increased to 1: 1.Stop after 84 hours cultivating the sampling and measuring saccharifying enzymic activity.
4. result
The work of IN7-31 proteinase of moldy bran enzyme is the bent powder of 8248 units/g (amount to definition 2 and be expressed as the bent powder of 137742 units/g), and the work of control group A s3.4309 proteinase of moldy bran enzyme is 2624 units/g song powder (amount to definition 2 and be expressed as the bent powder of 43820 units/g).
Embodiment 2 high glucoamylase enzymes wheat bran alive makes 1
1. bran mass
Wheat bran: W-Gum: water=100: 4: 100,
2. the preparation of high glucoamylase enzyme wheat bran alive
Inclined-plane twice the Aspergillus niger strain IN7-31 of activation that goes down to posterity is inoculated in the wheat bran seed culture medium respectively with certain inoculum size, cultivates for 28 ℃ and made the production bacterial classification in 72 hours.IN7-31 is produced bacterial classification be inoculated in bran mass with 10% inoculum size, place 28 ℃ of culturing room, ventilate from the false bottom of cylinder and cultivate, ventilation is 1: 0.5, cultivates and turns over Qu Yici after 48 hours, and ventilation was increased to 1: 1.Stop after 70 hours cultivating the sampling and measuring saccharifying enzymic activity.
3. result
The work of IN7-31 proteinase of moldy bran enzyme is the bent powder of 7540 units/g (amount to definition 2 and be expressed as the bent powder of 125918 units/g), and being scaled the work of over dry inulinase is the bent powder of 11712 units/g (amount to definition 2 and be expressed as the bent powder of 195590 units/g).
Embodiment 3 high glucoamylase enzymes wheat bran alive makes 2
1. bran mass
Wheat bran: ammonium sulfate: potassium primary phosphate: W-Gum: water ,=100: 0.5: 0.3: 3: 120
2. the preparation of high glucoamylase enzyme wheat bran alive
The preparation of producing bacterial classification is with embodiment 2.
IN7-31 is produced bacterial classification be inoculated in bran mass with 5% inoculum size, place 30 ℃ of culturing room, ventilate from the false bottom of cylinder and cultivate, ventilation is 1: 0.5, cultivates and turns over Qu Yici after 48 hours, and ventilation was increased to 1: 1.Stop after 90 hours cultivating the sampling and measuring saccharifying enzymic activity.
3. result
The work of IN7-31 proteinase of moldy bran enzyme is the bent powder of 8539 units/g (amount to definition 2 and be expressed as the bent powder of 142601 units/g).
Embodiment 4 utilizes high glucoamylase enzyme wheat bran alive to optimize Shanxi mature vinegar traditional fermentation technology
Control group 1 adopts traditional Shanxi mature vinegar wine-making technology, mixed Chinese sorghum, Daqu and water according to the rules, every cylinder charging 2kg, by processing requirement control room temperature at 25～28 ℃, fermentation period 18 days.Control group 2 is replaced half Daqu with As3.4309 and IN7-31 wheat bran respectively with the optimization group, and all the other processing parameters are with traditional production technique.Control group and optimization group are all implemented three parallel tests.
After the fermentation ends, adopt the ethanol content in the gas chromatographic detection fermented liquid, and measure the first sugar and the residual sugar of karusen.
The results are shown in following table:
| Wine-making technology | Just sugared (g/v) | Residual sugar (g/v) | Ethanol concn (v/v) | The sugar wine transformation efficiency | Starch utilization ratio |
| Control group 1 control group 2 optimization groups | 17.3％ 14.1％ 14.1％ | 6.7％ 4.0％ 3.1％ | 5％ 5.2％ 6.2％ | 37.7％ 41.2％ 45.1％ | 61.3％ 71.6％ 78.0％ |
Embodiment 5 shakes the liquid saccharified zymin of bottle preparation
1. substratum
The potato substratum: prescription does not add agar with embodiment 1.
The shake flask fermentation substratum: Semen Maydis powder 8g, bean powder 3g, ammonium sulfate 0.05g, wheat bran 2g, water 90ml places the 300ml triangular flask, 121 ℃, 20min sterilization.
2. the preparation of liquid saccharified enzyme
The production strain preparation: inclined-plane twice the Aspergillus niger strain IN7-31 of activation that goes down to posterity is inoculated in the potato substratum with certain inoculum size, shaking speed 120rpm, 28 ℃ of temperature are cultivated and were made the production bacterial classification in 48 hours.
The preparation of liquid saccharified enzyme: IN7-31 is produced bacterial classification be inoculated in respectively in the shake flask fermentation substratum, place 28 ℃ of shaker fermentations to cultivate shaking speed 120rpm with 5% inoculum size.Stop after 96 hours cultivating the sampling and measuring saccharifying enzymic activity.
3. result
The work of IN7-31 shake flask fermentation thinning enzyme enzyme is 2256 units/ml fermented liquid (amount to definition 2 and be expressed as 37675 units/ml fermented liquid).
Embodiment 6 small-sized fermentation jars prepare liquid saccharified zymin
1. substratum
Liquid bacterium culture medium: potato substratum (prescription is with embodiment 5)
Fermention medium: Semen Maydis powder 60g, Zulkovsky starch 20g, soybean cake powder 10g, ammonium sulfate 1.5g, wheat bran 10g, tap water 900ml.121 ℃, 40min sterilization.
2. the preparation of liquid saccharified zymin
Strain preparation: get one on fresh IN7-31 inclined-plane, wash spore with sterilized water, be inoculated in the liquid bacterium culture medium, 28 ℃, 120rpm shaking table are cultivated 48h and are promptly made liquid bacterial classification.
Liquid submerged aerobic fermentation prepares saccharifying enzyme: adopt 5 liters of automatic fermenters to test, substratum is a fermention medium, coefficient is 70%, inoculum size is 10%, pH is controlled to be 6, and temperature is controlled to be 28 ℃, and dissolved oxygen is controlled to be 20%, no-feed supplement in the fermenting process, fermenting, it is alive to measure the fermented liquid glucoamylase enzyme after 92 hours.
3. result
The fermented liquid glucoamylase enzyme work of IN7-31 is 3531 units/ml fermented liquid (amount to definition 2 and be expressed as 58967 units/ml fermented liquid).
Embodiment 7 small-sized fermentation jars prepare liquid saccharified zymin
1. substratum
Liquid bacterium culture medium: potato substratum (prescription is with embodiment 5)
Fermention medium: Semen Maydis powder 100g, soybean cake powder 10g, ammonium sulfate 3g, dipotassium hydrogen phosphate 1.5g, potassium primary phosphate 1.5g, wheat bran 10g, tap water 900ml.121 ℃, 40min sterilization.
2. the preparation of liquid saccharified zymin
Strain preparation: with embodiment 6 strain preparation.
Liquid submerged aerobic fermentation fermentative preparation saccharifying enzyme: adopt 5 liters of automatic fermenters to test, substratum is a fermention medium, coefficient is 70%, inoculum size is 10%, pH is controlled to be 6, and temperature is controlled to be 28 ℃, and dissolved oxygen is controlled to be 20%, no-feed supplement in the fermenting process, fermenting, it is alive to measure the fermented liquid glucoamylase enzyme after 65 hours.
3. result
The fermented liquid glucoamylase enzyme work of IN7-31 is 4120 units/ml fermented liquid (amount to definition 2 and be expressed as 68804 units/ml fermented liquid).
Claims (10)
1, a kind of high saccharifying enzyme bacterial classification alive is characterized in that described bacterial classification is an aspergillus niger IN7-31 bacterial strain, and preserving number is CGMCCNo.1818.
2, a kind of high saccharifying enzyme wheat bran of living, it is characterized in that adopting bran mass and preserving number is that the spawn culture of CGMCC No.1818 makes.
3,, it is characterized in that comprising the steps: that (1) is that the bacterial classification of CGMCC No.1818 is slant medium activation 2～4 times with preserving number according to the live preparation method of wheat bran of the described a kind of high saccharifying enzyme of claim 2; (2) the activatory bacterial classification is inserted the wheat bran seed culture medium, under 25～33 ℃ of temperature, cultivate and made solid state bacterial in 24～96 hours; (3) solid state bacterial is inserted in the bran mass, ventilating to cultivating under 25～33 ℃ of temperature promptly made high saccharifying enzyme wheat bran alive in 70～100 hours.
4, according to the preparation method of the described a kind of high saccharifying enzyme of claim 3 wheat bran alive, it is characterized in that culture temperature is 28～30 ℃ in described step (2), (3).
5, according to the live purposes of wheat bran of the described a kind of high saccharifying enzyme of claim 2, in wine brewing, make the application in producing of vinegar, alcohol, organic acid.
6, a kind of high saccharifying enzyme liquid formulation of living, it is characterized in that adopting liquid state fermentation substratum and preserving number is that the spawn culture of CGMCCNo.1818 makes.
7,, it is characterized in that comprising the steps: that (1) is that the bacterial classification of CGMCC No.1818 is slant medium activation 2～4 times with preserving number according to the live preparation method of liquid formulation of the described a kind of high saccharifying enzyme of claim 6; (2) the activatory bacterial classification is inserted liquid bacterium culture medium, under 25～33 ℃ of temperature, cultivate and made liquid bacterial classification in 24～72 hours; (3) liquid bacterial classification is inserted in the liquid state fermentation substratum, control pH is 5～7, and under 25～33 ℃ of temperature, ventilation, stir culture promptly made high saccharifying enzyme liquid formulation alive in 50～100 hours.
8, according to the preparation method of the described a kind of high saccharifying enzyme of claim 7 liquid formulation alive, it is characterized in that culture temperature is 28～30 ℃ in described step (2), (3).
9, according to the preparation method of the described a kind of high saccharifying enzyme of claim 7 liquid formulation alive, it is characterized in that pH is 6 in the described step (3).
10, according to the purposes of the described a kind of high saccharifying enzyme of claim 6 liquid formulation alive, it is characterized in that being used to prepare refining saccharifying enzyme, and the application in wine brewing, wine vinegar and alcohol, organic acid, production of antibiotics.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255741A (en) * | 2015-10-19 | 2016-01-20 | 山东隆科特酶制剂有限公司 | Aspergillus niger mutant strain with high yield of glucoamylase and industrial fermentation technology of aspergillus niger mutant strain |
| CN107857605A (en) * | 2017-11-08 | 2018-03-30 | 贵州省化工研究院 | One kind polymerization ureaformaldehyde method of modifying and modified poly ureaformaldehyde and its application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1210888A (en) * | 1998-10-05 | 1999-03-17 | 清华大学 | Process for producing saccharifying enzyme using waste molasses as main raw material |
| CN100443589C (en) * | 2005-12-30 | 2008-12-17 | 湖南鸿鹰祥生物工程股份有限公司 | Production of beta-amylase concentrated liquid with high conversion-rate and purity |
-
2006
- 2006-10-20 CN CNB2006101020563A patent/CN100427583C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255741A (en) * | 2015-10-19 | 2016-01-20 | 山东隆科特酶制剂有限公司 | Aspergillus niger mutant strain with high yield of glucoamylase and industrial fermentation technology of aspergillus niger mutant strain |
| CN105255741B (en) * | 2015-10-19 | 2018-12-07 | 山东隆科特酶制剂有限公司 | The aspergillus niger mutant strain of one plant of high-yield glucoamylase and its industrial fermentation technology |
| CN107857605A (en) * | 2017-11-08 | 2018-03-30 | 贵州省化工研究院 | One kind polymerization ureaformaldehyde method of modifying and modified poly ureaformaldehyde and its application |
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