CN102876646A - Method for producing xylanase by fermentation of koji tray of Aspergillus niger and culture medium used by method - Google Patents
Method for producing xylanase by fermentation of koji tray of Aspergillus niger and culture medium used by method Download PDFInfo
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Abstract
The invention discloses a method for producing xylanase by the fermentation of a koji tray of Aspergillus niger and a culture medium used by the method, and belongs to the technical field of bioengineering. According to the method, cheap agricultural byproducts such as bran, corncobs and bean cake powder are used as a fermentation base material, a proper amount of inorganic nitrogen source, surfactant and inorganic salt are added, and the xylanase is produced by utilizing the fermentation of a koji tray of an Aspergillus niger XZ-3S strain. A formula and culture conditions of a koji tray fermentation culture medium for producing the xylanase by using the Aspergillus niger XZ-3S strain in a laboratory are provided; and the activity of acidic xylanase of mature fermentation koji of the culture medium is up to 23,065 to 26,380IU/g (dry koji), and is higher than the current domestic level. The invention has the advantages that equipment is simple, the method is low in investment, quick in response and low in production cost, and is environment-friendly, the enzyme activity of solid-state fermentation of the xylanase is relatively high, and the like; and the method is favorable for the development and utilization of the xylanase, and has relatively high economic and practical value.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of method and used medium thereof of being produced zytase by the aspergillus niger koji plate fermentation, particularly refer to utilize aspergillus niger (Aspergillus niger) XZ-3S bacterial strain koji plate fermentation to produce the novel process of zytase.
Background technology
Xylan is a kind of heterozygosis poly molecule, and main chain is linked to each other by the wood sugar glycosidic bond by a plurality of xylopyranosyl.According to the type of glycosidic link, xylan can be divided into two kinds, and β-Isosorbide-5-Nitrae-xylan and β-1, the 3-xylan.The former is present in the terrestrial plant cell walls, and its main chain is to be polymerized by the continuous β of β-Isosorbide-5-Nitrae-glycosidic link-D-xylopyranose residue; And the latter is present in the cell walls of marine algae, and by β-1, the 3-glycosidic link is polymerized its main chain by β-D-xylopyranose residue.The xylan of occurring in nature mostly is different poly-polysaccharide; the short substituting group of the multiple different sizes of ining succession on the side chain mainly contains O-ethanoyl, 4-O-methyl D-glucuronic acid residue, L-arabinose residue (can further link to each other with phenolic acid such as coumaric acid, forulic acid) etc.Other several structural polysaccharides (such as xylogen, Mierocrystalline cellulose, pectin, dextran etc.) are connected with key covalently or non-covalently in these side chains and the vegetable cell, form the important structure-cell walls of vegetable cell.
Zytase (Xylanases) is that a class plays synergistic polycomponent enzyme system in the xylan degrading process, belong to the hemicellulose enzyme, be often referred to β-1,4-D-endoxylanase (β-1,4-D-endoxylanase, EC3.2.1.8), can from the inside hydrolysis xyloside key of xylan backbone, it be degraded into oligomeric xylose, xylo-bioses and a small amount of wood sugar.Zytase has broad application prospects in numerous industries such as food, animal-feed, papermaking, and the research zytase has good commercial value and scientific meaning.
Zytase is very wide in distributed in nature, all has zytase in ocean and land bacterium, marine algae, fungi, yeast, cud and ruminating animal bacterium, snail, crustacean, land plant tissue and various invertebrates.Studying more is the zytase of microorganisms, has reported that the microorganism that can synthesize zytase has bacterium, actinomycetes, fungi and yeasts etc.At present research the most deeply, what be most widely used is that aspergillus, mould and wood in the fungi is mould, and their zytases that produces mostly are acidity or neutral enzymatic.In recent years, people screen some thermoduric bacterias or alkaline-resisting bacterium from some extreme environments, and the thermotolerance that it produces or alkali resistance zytase have caused people's concern.Simultaneously, the xylanase gene engineering strain that some is artificial constructed, xylanase activity is high because it produces, enzyme is application requiring pure, that zymologic property meets people's setting, also is widely applied.
Hou Bainan etc. utilize aspergillus niger (Aspergillus niger) N86 bacterial strain, and zytase is produced in liquid state fermentation, and enzyme activity reaches 139IU/mL; It is the 4915IU/g dry medium that the aobvious good grade of journey is utilized bacillus pumilus, Produced by Solid-state Fermentation xylanase activity; Fu Dandan etc. utilize Aspergillus usamii E001 bacterial strain, and the Produced by Solid-state Fermentation xylanase activity is 7442IU/g.Wu Minchen etc. are in patent of invention " a kind of method of utilizing the Aspergillus usamii industrialization to produce zytase ", reported the method for utilizing the Aspergillus usamii industrialization to produce zytase among the publication number CN101067129, zytase solid state fermentation enzymic activity is 7783～9616IU/g dry medium.
At present, no matter be solution fermentation or solid state fermentation, the production by biological xylanase activity is all lower, has caused the production of zytase and application cost high, thereby has limited the widespread use of this enzyme; And have no the document or the patent report that utilize the aspergillus niger koji plate fermentation to produce zytase.The enzymic activity that the present invention utilizes the aspergillus niger koji plate fermentation to produce zytase is higher than present domestic level.
Summary of the invention
The object of the invention is to disclose a kind of method of being produced zytase by the aspergillus niger koji plate fermentation.
Second purpose of the present invention is to disclose by the aspergillus niger koji plate fermentation produces the employed substratum of zytase.
The objective of the invention is to be achieved through the following technical solutions:
A kind of method of being produced zytase by the aspergillus niger koji plate fermentation, wherein, described method is take aspergillus niger XZ-3S as raw material, carries out solid state fermentation at the koji plate fermentation substratum, obtain behind the leaven material leaven material to be extracted with damping fluid, obtain zytase liquid; Consisting of of described koji plate fermentation substratum
Koji tray dress 100g base-material, (NH
4)
2SO
40.5～1.5g, peptone 0.5～1g, KH
2PO
40.3～0.4g, CaCl
20.2～0.3g, ZnCl
20.1～0.2g, MgSO
40.1～0.2g, Fe
2(SO
4)
30.02～0.08g, polysorbate40 0.4～0.8g, tap water 120～140mL, natural pH, wherein base-material consists of wheat bran 50～80g, corn cob 10～40g, soybean cake powder 10～20g.
The described method of being produced zytase by the aspergillus niger koji plate fermentation of technique scheme, wherein, the condition of described solid fermentation is 121 ℃ of sterilizations of koji plate fermentation substratum 40min, inoculation 10mL aspergillus niger XZ-3S wheat bran spore suspension after the cooling, and this suspension miospore concentration is 5 * 10
7Individual/mL, 29 ℃ leave standstill and cultivate 65～72h, during respectively turn over Qu Yici during respectively at 24～28h and 43～47h moisturizing 20mL.
The described method of being produced zytase by the aspergillus niger koji plate fermentation of technique scheme, wherein, described damping fluid is 0.05mol/L Sodium phosphate dibasic-citrate buffer solution.
The described method of being produced zytase by the aspergillus niger koji plate fermentation of technique scheme, wherein, the process that the leaven material is extracted with damping fluid is for taking by weighing leaven material 1g, add 29mL pH4.0,0.05mol/L Sodium phosphate dibasic-citrate buffer solution, lixiviate 30min under 40 ℃ of conditions, filter paper filtering, gained filtrate is zytase liquid.
A kind of method of being produced zytase by the aspergillus niger koji plate fermentation comprises the steps:
(1), inclined-plane seed culture: peeling potato 200g/L, sucrose 20g/L, agar 20g/L, pH6.0; 121 ℃ of sterilization 20min, the holding test tubes inclined-plane; Inoculated aspergillus niger XZ-3S bacterial strain after the cooling is cultivated 96h for 30 ℃;
(2), wheat bran seed preparation: the bottled 10g base-material of 250mL triangle, (NH
4)
2SO
480mg, tap water 10mL, pH6.0, abundant mixing, 121 ℃ of sterilization 40min, the aspergillus niger XZ-3S test tube slant seed of inoculation step (1) gained after the cooling, 30 ℃ leave standstill and cultivate 96h, during respectively turn over Qu Yici respectively at 24h and 44h, described base-material is wheat bran;
(3), wheat bran spore suspension preparation: the bottled 20mL deionized water of 100mL triangle, put into 10-15 grain granulated glass sphere, 121 ℃ of sterilization 20min, after placing room temperature, access 1 inoculation shovel wheat bran seed is used first the granulated glass sphere vibrating dispersion, and then filter with absorbent cotton, adjusting spore concentration is 5 * 10
7Individual/mL;
(4), koji plate fermentation: 121 ℃ of koji plate fermentation substratum sterilization 40min, inoculation 10mL aspergillus niger XZ-3S wheat bran spore suspension after the cooling, this suspension miospore concentration is 5 * 10
7Individual/mL, 29 ℃ leave standstill and cultivate 65～72h, during respectively turn over Qu Yici during respectively at 24～28h and 43～47h moisturizing 20mL; Consisting of of described koji plate fermentation substratum
Koji tray dress 100g base-material, (NH
4)
2SO
40.5～1.5g, peptone 0.5～1g, KH
2PO
40.3～0.4g, CaCl
20.2～0.3g, ZnCl
20.1～0.2g, MgSO
40.1～0.2g, Fe
2(SO
4)
30.02～0.08g, polysorbate40 0.4～0.8g, tap water 120～140mL, natural pH, wherein base-material consists of wheat bran 50～80g, corn cob 10～40g, soybean cake powder 10～20g;
(5), crude enzyme liquid extracts: koji plate fermentation takes by weighing leaven material 1g after finishing, and adds 29mL pH4.0,0.05mol/L Sodium phosphate dibasic-citrate buffer solution, lixiviate 30min under 40 ℃ of conditions, filter paper filtering, gained filtrate is zytase liquid.
The described a kind of method of being produced zytase by the aspergillus niger koji plate fermentation of technique scheme, wherein, the consisting of of described koji plate fermentation substratum
Koji tray dress 100g base-material, (NH
4)
2SO
4Lg, peptone 0.5g, KH
2PO
40.3g, CaCl
20.2g, ZnCl
20.1g, MgSO
40.1, Fe
2(SO
4)
30.02g, polysorbate40 0.4g, tap water 140mL, natural pH, wherein base-material consists of wheat bran 50g, corn cob 40g, soybean cake powder 10g.
The described a kind of employed koji plate fermentation substratum of method of being produced zytase by the aspergillus niger koji plate fermentation of above-mentioned arbitrary technical scheme, wherein, the consisting of of described koji plate fermentation substratum
Koji tray dress 100g base-material, (NH
4)
2SO
40.5～1.5g, peptone 0.5～1g, KH
2PO
40.3～0.4g, CaCl
20.2～0.3g, ZnCl
20.1～0.2g, MgSO
40.1～0.2g, Fe
2(SO
4)
30.02～0.08g, polysorbate40 0.4～0.8g, tap water 120～140mL, natural pH, wherein base-material consists of wheat bran 50～80g, corn cob 10～40g, soybean cake powder 10～20g.
The described koji plate fermentation substratum of technique scheme, wherein, the φ 20cm * 4cm koji tray that consists of of described koji plate fermentation substratum fills 100g base-material, (NH
4)
2SO
41g, peptone 0.5g, KH
2PO
40.3g, CaCl
20.2g, ZnCl
20.1g, MgSO
40.1, Fe
2(SO
4)
30.02g, polysorbate40 0.4g, tap water 140mL, natural pH, wherein base-material consists of wheat bran 50g, corn cob 40g, soybean cake powder 10g.
The described koji plate fermentation substratum of technique scheme, wherein, the φ 20cm * 4cm koji tray that consists of of described koji plate fermentation substratum fills 100g base-material, (NH
4)
2SO
41g, peptone 0.5g, KH
2PO
40.3g, CaCl
20.2g, ZnCl
20.1g, MgSO
40.1g, Fe
2(SO
4)
30.02g, polysorbate40 0.4g, tap water 140mL, natural pH, wherein base-material consists of wheat bran 80g, corn cob 10g, soybean cake powder 10g
The described koji plate fermentation substratum of technique scheme, wherein, the φ 20cm * 4cm koji tray that consists of of described koji plate fermentation substratum fills 100g base-material, (NH
4)
2SO
41g, peptone 0.5g, KH
2PO
40.3g, CaCl
20.2g, ZnCl
20.1g, MgSO
40.1g, Fe
2(SO
4)
30.02g, polysorbate40 0.4g, tap water 120mL, natural pH, wherein base-material consists of wheat bran 80g, corn cob 10g, soybean cake powder 10g.
The purpose of this invention is to provide a kind ofly take agricultural byproducts such as wheat bran, corn cob, soybean cake powder as the fermentation base-material, utilize aspergillus niger XZ-3S koji plate fermentation to produce the method for zytase.The technical scheme of koji plate fermentation of the present invention and theing contents are as follows:
1, bacterial strain: aspergillus niger (Aspergillus niger) XZ-3S bacterial strain, by Xinxiang College of Medical Science's screening and preservation, this bacterial strain is open at the Vol.33No.16Pg.187 of " foodstuffs industry science and technology " magazine in 2012, the inventor and applicant promise to undertake that this bacterial strain provides to the public in 20 years from the applying date, the public can obtain by the fermentation of Life Sci-Tech institute of contact Xinxiang College of Medical Science and enzyme engineering research department the bacterial classification of freeze-drying preservation.
The mycology feature of aspergillus niger XZ-3S bacterial strain is as follows: aspergillus niger XZ-3S bacterial strain belongs to the Deuteromycotina fungi; Bacterium colony is white just, rear blackening; The conidial head brown-black is radial, and conidiophore is different in size; The top capsule is spherical, double-deck stigma; The conidium brown is spherical; 37 ℃ of growth thermophilics, minimum relative humidity is 88%; Aspergillus niger has in the gathering of culturing process miospore or sprouts into mycelia, and mycelia is twined the feature of balling-up mutually.
2, inclined-plane seed culture: peeling potato 200g/L, sucrose 20g/L, agar 20g/L, pH6.0; 121 ℃ of sterilization 20min, the holding test tubes inclined-plane; Inoculated aspergillus niger XZ-3S bacterial strain after the cooling is cultivated 96h for 30 ℃.This slant medium can be used for activation, preservation and the inclined-plane seed etc. of bacterial classification, switching in per 3 months 1 time.
3, wheat bran seed preparation: the bottled 10g base-material of 250mL triangle (wheat bran 10g), (NH
4)
2SO
480mg, tap water 10mL, pH6.0, abundant mixing, 121 ℃ of sterilization 40min, inoculated aspergillus niger XZ-3S test tube slant seed after the cooling, 30 ℃ leave standstill and cultivate 96h, during respectively turn over Qu Yici respectively at 24h and 44h.
4, wheat bran spore suspension preparation: the bottled 20mL deionized water of 100mL triangle, put into 10-15 grain granulated glass sphere, 121 ℃ of sterilization 20min, after placing room temperature, access 1 inoculation shovel wheat bran seed is used first the granulated glass sphere vibrating dispersion, and then filter with absorbent cotton, adjusting spore concentration is 5 * 10
7Individual/mL.
5, koji plate fermentation: koji tray (dress of big or small φ 20cm * 4cm) 100g base-material (wheat bran 50～80g, corn cob 10～40g, soybean cake powder 10～20g), (NH
4)
2SO
40.5～1.5g, peptone 0.5～1g, KH
2PO
40.3～0.4g, CaCl
20.2～0.3g, ZnCl
20.1～0.2g, MgSO
40.1～0.2g, Fe
2(SO
4)
30.02～0.08g, polysorbate40 0.4～0.8g, tap water 120～140mL, natural pH.121 ℃ of sterilization 40min, (spore concentration is 5 * 10 to inoculation 10mL aspergillus niger XZ-3S wheat bran spore suspension after the cooling
7Individual/mL), 29 ℃ leave standstill and cultivate 65～72h, during respectively turn over Qu Yici during respectively at 24～28h and the normal moisturizing 20mL of 43～47h.
6, thick enzyme (zytase) liquid extracts: solid state fermentation takes by weighing leaven material 1g after finishing, add 29mL pH4.0,0.05mol/L Sodium phosphate dibasic-citrate buffer solution, lixiviate 30min under 40 ℃ of conditions, filter paper filtering, gained filtrate is zytase liquid.
7, analytical procedure:
(1), xylanase activity is measured: in 25mL tool plug test tube A and B, add respectively the mass concentration of preparing with pH4.0, Sodium phosphate dibasic-citrate buffer solution and be 0.5% xylan solution 2.4mL, 45 ℃ of preheating 5～10min, in the A pipe, add the suitably enzyme liquid of dilution of 0.1mL, 45 ℃ of accurate response 15min; Respectively add immediately 2.5mL3 ', 5 '-dinitrosalicylic acid (DNS) reagent, in the B pipe, add again 0.1mL enzyme liquid, A and B pipe all boil 7min; Respectively add deionized water 5mL, mixing after the cooling; 540nm sentences the B pipe and measures A pipe absorbance for blank, and finds corresponding reducing sugar (in wood sugar) content and be converted to unit of enzyme activity from the wood sugar typical curve.Unit of enzyme activity is defined as: under this condition determination, produce the required enzyme amount of 1 μ mol reducing sugar with per minute and be defined as 1 xylanase activity unit (IU).
(2), reducing sugar test: adopt the DNS method, take wood sugar as standard.
(3), bent material moisture determination (wet Qu Ganchong measures): take by weighing a certain amount of solid state fermentation culture (bent material), dry to constant weight for 105 ℃.
Analytical procedure of the present invention (1) is enzyme activity determination, and the absorbance of mensuration needs to find corresponding reducing sugar (in wood sugar) content and be converted to unit of enzyme activity from the wood sugar typical curve; (2) reducing sugar test is the method for wood sugar typical curve preparation.The enzyme of (1) mensuration is lived and is lived for the enzyme in the crude enzyme liquid in addition, and unit is IU/mL; Because the present invention is the final enzyme of solid state fermentation to live and is the IU/g dry medium, is converted to the IU/g dry medium from IU/mL and need to measures bent material moisture (3).
The present invention has following beneficial effect:
(1), the agricultural byproducts such as cheap wheat bran, corn cob that utilize of the present invention are as the fermentation main raw material, and the production bacterial classification aspergillus niger (Aspergillus niger) that adopts is the microorganism of the safety (GRAS) of generally acknowledging, it has safety, reliably and not produces the characteristics such as toxin.
(2), the present invention utilizes aspergillus niger XZ-3S bacterial strain koji plate fermentation to produce zytase to be a kind of nature renewable resources, environmental protection and continuable mode of production of taking full advantage of, to have simultaneously the advantages such as equipment is simple, less investment, instant effect, low production cost.
(3), the present invention the laboratory on a small scale on, utilize aspergillus niger XZ-3S bacterial strain koji plate fermentation to produce zytase, its zytase fermenting enzyme activity can reach 23065～26380IU/g dry medium.
Embodiment:
For making technical scheme of the present invention be convenient to understand, the present invention is further illustrated below in conjunction with concrete test example.
Embodiment 1:
1. bacterial strain: aspergillus niger (Aspergillus niger) XZ-3S bacterial strain.
2, inclined-plane seed culture: peeling potato 200g/L, sucrose 20g/L, agar 20g/L, pH6.0; 121 ℃ of sterilization 20min, the holding test tubes inclined-plane; Inoculated aspergillus niger XZ-3S bacterial strain after the cooling is cultivated 96h for 30 ℃;
3, wheat bran seed preparation: the bottled 10g base-material of 250mL triangle, (NH
4)
2SO
480mg, tap water 10mL, pH6.0, abundant mixing, 121 ℃ of sterilization 40min, the aspergillus niger XZ-3S test tube slant seed of inoculation step (1) gained after the cooling, 30 ℃ leave standstill and cultivate 96h, during respectively turn over Qu Yici respectively at 24h and 44h, described base-material is wheat bran;
4, wheat bran spore suspension preparation: the bottled 20mL deionized water of 100mL triangle, put into 10-15 grain granulated glass sphere, 121 ℃ of sterilization 20min, after placing room temperature, access 1 inoculation shovel wheat bran seed is used first the granulated glass sphere vibrating dispersion, and then filter with absorbent cotton, adjusting spore concentration is 5 * 10
7Individual/mL;
5. solid-state fermentation culture medium: koji tray (the dress 100g base-material (wheat bran 50g, corn cob 40g, soybean cake powder 10g) of big or small φ 20cm * 4cm), (NH
4)
2SO
4Lg, peptone 0.5g, KH
2PO
40.3g, CaCl
20.2g, ZnCl
20.1g, MgSO
40.1g, Fe
2(SO
4)
30.02g, polysorbate40 0.4g, tap water 140mL, natural pH.
6. solid state fermentation conditions: 121 ℃ of sterilization 40min, (spore concentration is 5 * 10 to inoculation 10mL aspergillus niger XZ-3S wheat bran spore suspension after the cooling
7Individual/mL), 29 ℃ leave standstill and cultivate 65h, during respectively turn over Qu Yici during respectively at 28h and the normal moisturizing of 47h (20mL).
The above-mentioned koji tray experimental result that repeats 3 batches (every batches parallel do 3 koji traies) shows, the average enzyme of the ripe solid state fermentation of XZ-3S bent material crude enzyme liquid is lived and is 12398.6IU/mL, wet Qu Pingjun dry weight is that 0.47g dry medium/g is wet bent, calculates the average enzymic activity of bent material zytase and reaches the 26380IU/g dry medium.
Embodiment 2:
The present embodiment and embodiment are basic identical, and difference is on solid-state fermentation culture medium and the solid state fermentation conditions, and is concrete:
5, solid-state fermentation culture medium: koji tray (dress of big or small φ 20cm * 4cm) 100g base-material (wheat bran 80g, corn cob 10g, soybean cake powder 10g), (NH
4)
2SO
41g, peptone 0.5g, KH
2PO
40.3g, CaCl
20.2g, ZnCl
20.1g, MgSO
40.1g, Fe
2(SO
4)
30.02g, polysorbate40 0.4g, tap water 140mL, natural pH.
6, solid state fermentation conditions: 121 ℃ of sterilization 40min, (spore concentration is 5 * 10 to inoculation 10mL aspergillus niger XZ-3S wheat bran spore suspension after the cooling
7Individual/mL), 29 ℃ leave standstill and cultivate 65h, during respectively turn over Qu Yici during respectively at 28h and the normal moisturizing of 47h (20mL).
The above-mentioned koji tray experimental result that repeats 3 batches (every batches parallel do 3 koji traies) shows, the average enzyme of the ripe solid state fermentation of XZ-3S bent material crude enzyme liquid is lived and is 10829.25IU/mL, wet Qu Pingjun dry weight is that 0.45g dry medium/g is wet bent, and the average enzymic activity of the zytase of calculating reaches the 24065IU/g dry medium.
Embodiment 3:
The present embodiment and embodiment are basic identical, and difference is on solid-state fermentation culture medium and the solid state fermentation conditions, and is concrete:
5, solid-state fermentation culture medium: the koji tray (dress of big or small φ 20cm * 4cm) 100g base-material (wheat bran 80g, corn cob 10g, soybean cake powder 10g) (NH
4)
2SO
41g, peptone 0.5g, KH
2PO
40.3g, CaCl
20.2g, ZnCl
20.1g, MgSO
40.1g, Fe
2(SO
4)
30.02g, polysorbate40 0.4g, tap water 120mL, natural pH
6, solid state fermentation conditions: 121 ℃ of sterilization 40min, (spore concentration is 5 * 10 to inoculation 10mL aspergillus niger XZ-3S wheat bran spore suspension after the cooling
7Individual/mL), 29 ℃ leave standstill and cultivate 65h, during respectively turn over Qu Yici during respectively at 28h and the normal moisturizing of 47h (20mL).
The above-mentioned koji tray experimental result that repeats 3 batches (every batches parallel do 3 koji traies) shows, the average enzyme of the ripe solid state fermentation of XZ-3S bent material crude enzyme liquid is lived and is 8995.35IU/mL, wet Qu Pingjun dry weight is that 0.39g dry medium/g is wet bent, and the average enzymic activity of the zytase of calculating reaches the 23065IU/g dry medium.
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any formal and substantial restriction, all those skilled in the art, within not breaking away from the technical solution of the present invention scope, when can utilizing the disclosed above technology contents, and a little change of making, modify the equivalent variations with differentiation, be equivalent embodiment of the present invention; Simultaneously, the change of any equivalent variations that all foundations essence technology of the present invention is done above embodiment, modify and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (10)
1. method of being produced zytase by the aspergillus niger koji plate fermentation, it is characterized in that: described method is as raw material take aspergillus niger XZ-3S, carry out solid state fermentation at the koji plate fermentation substratum, obtain behind the leaven material leaven material to be extracted with damping fluid, obtain zytase liquid; Consisting of of described koji plate fermentation substratum
Koji tray dress 100g base-material, (NH
4)
2SO
40.5～1.5g, peptone 0.5～1g, KH
2PO
40.3～0.4g, CaCl
20.2～0.3g, ZnCl
20.1～0.2g, MgSO
40.1～0.2g, Fe
2(SO
4)
30.02～0.08g, polysorbate40 0.4～0.8g, tap water 120～140mL, natural pH, wherein base-material consists of wheat bran 50～80g, corn cob 10～40g, soybean cake powder 10～20g.
2. method of being produced zytase by the aspergillus niger koji plate fermentation according to claim 1, it is characterized in that: the condition of described solid fermentation is 121 ℃ of sterilizations of koji plate fermentation substratum 40min, inoculation 10mL aspergillus niger XZ-3S wheat bran spore suspension after the cooling, this suspension miospore concentration is 5 * 10
7Individual/mL, 29 ℃ leave standstill and cultivate 65～72h, during respectively turn over Qu Yici during respectively at 24～28h and 43～47h moisturizing 20mL.
3. method of being produced zytase by the aspergillus niger koji plate fermentation according to claim 1, it is characterized in that: described damping fluid is 0.05mol/L Sodium phosphate dibasic-citrate buffer solution.
4. the described method of arbitrary claim according to claim 1～3, it is characterized in that: the process that the leaven material is extracted with damping fluid is for taking by weighing leaven material 1g, add 29mL pH4.0,0.05mol/L Sodium phosphate dibasic-citrate buffer solution, lixiviate 30min under 40 ℃ of conditions, filter paper filtering, gained filtrate is zytase liquid.
5. a method of being produced zytase by the aspergillus niger koji plate fermentation comprises the steps:
(1), inclined-plane seed culture: peeling potato 200g/L, sucrose 20g/L, agar 20g/L, pH6.0; 121 ℃ of sterilization 20min, the holding test tubes inclined-plane; Inoculated aspergillus niger XZ-3S bacterial strain after the cooling is cultivated 96h for 30 ℃;
(2), wheat bran seed preparation: the bottled 10g base-material of 250mL triangle, (NH
4)
2SO
480mg, tap water 10mL, pH6.0, abundant mixing, 121 ℃ of sterilization 40min, the aspergillus niger XZ-3S test tube slant seed of inoculation step (1) gained after the cooling, 30 ℃ leave standstill and cultivate 96h, during respectively turn over Qu Yici respectively at 24h and 44h, described base-material is wheat bran;
(3), wheat bran spore suspension preparation: the bottled 20mL deionized water of 100mL triangle, put into 10-15 grain granulated glass sphere, 121 ℃ of sterilization 20min, after placing room temperature, access 1 inoculation shovel wheat bran seed is used first the granulated glass sphere vibrating dispersion, and then filter with absorbent cotton, adjusting spore concentration is 5 * 10
7Individual/mL;
(4), koji plate fermentation: 121 ℃ of koji plate fermentation substratum sterilization 40min, inoculation 10mL aspergillus niger XZ-3S wheat bran spore suspension after the cooling, this suspension miospore concentration is 5 * 10
7Individual/mL, 29 ℃ leave standstill and cultivate 65～72h, during respectively turn over Qu Yici during respectively at 24～28h and 43～47h moisturizing 20mL; Consisting of of described koji plate fermentation substratum
Cm koji tray dress 100g base-material, (NH
4)
2SO
40.5～1.5g, peptone 0.5～1g, KH
2PO
40.3～0.4g, CaCl
20.2～0.3g, ZnCl
20.1～0.2g, MgSO
40.1～0.2g, Fe
2(SO
4)
30.02～0.08g, polysorbate40 0.4～0.8g, tap water 120～140mL, natural pH, wherein base-material consists of wheat bran 50～80g, corn cob 10～40g, soybean cake powder 10～20g;
(5), crude enzyme liquid extracts: koji plate fermentation takes by weighing leaven material 1g after finishing, and adds 29mL pH4.0,0.05mol/L Sodium phosphate dibasic-citrate buffer solution, lixiviate 30min under 40 ℃ of conditions, filter paper filtering, gained filtrate is zytase liquid.
6. a kind of method of being produced zytase by the aspergillus niger koji plate fermentation according to claim 5 is characterized in that: the consisting of of described koji plate fermentation substratum
Koji tray dress 100g base-material, (NH
4)
2SO
4Lg, peptone 0.5g, KH
2PO
40.3g, CaCl
20.2g, ZnCl
20.1g, MgSO
40.1, Fe
2(SO
4)
30.02g, polysorbate40 0.4
g, tap water 140mL, natural pH, wherein base-material consists of wheat bran 50g, corn cob 40g, soybean cake powder 10g.
7. the described a kind of employed koji plate fermentation substratum of method of being produced zytase by the aspergillus niger koji plate fermentation of arbitrary claim among the claim 1-6 is characterized in that: the consisting of of described koji plate fermentation substratum
Koji tray dress 100g base-material, (NH
4)
2SO
40.5～1.5g, peptone 0.5～1g, KH
2PO
40.3～0.4g, CaCl
20.2～0.3g, ZnCl
20.1～0.2g, MgSO
40.1～0.2g, Fe
2(SO
4)
30.02～0.08g, polysorbate40 0.4～0.8g, tap water 120～140mL, natural pH, wherein base-material consists of wheat bran 50～80g, corn cob 10～40g, soybean cake powder 10～20g.
8. koji plate fermentation substratum according to claim 7 is characterized in that: described koji plate fermentation substratum consist of φ 20cm * 4cm koji tray dress 100g base-material, (NH
4)
2SO
4Lg, peptone 0.5g, KH
2PO
40.3g, CaCl
20.2g, ZnCl
20.1g, MgSO
40.1, Fe
2(SO
4)
30.02g, polysorbate40 0.4g, tap water 140mL, natural pH, wherein base-material consists of wheat bran 50g, corn cob 40g, soybean cake powder 10g.
9. koji plate fermentation substratum according to claim 7 is characterized in that: described koji plate fermentation substratum consist of φ 20cm * 4cm koji tray dress 100g base-material, (NH
4)
2SO
41g, peptone 0.5g, KH
2PO
40.3g, CaCl
20.2g, ZnCl
20.1g, MgSO
40.1g, Fe
2(SO
4)
30.02g, polysorbate40 0.4g, tap water 140mL, natural pH, wherein base-material consists of wheat bran 80g, corn cob 10g, soybean cake powder 10g.
10. koji plate fermentation substratum according to claim 7 is characterized in that: described koji plate fermentation substratum consist of φ 20cm * 4cm koji tray dress 100g base-material, (NH
4)
2SO
41g, peptone 0.5g, KH
2PO
40.3g, CaCl
20.2g, ZnCl
20.1g, MgSO
40.1g, Fe
2(SO
4)
30.02g, polysorbate40 0.4g, tap water 120mL, natural pH, wherein base-material consists of wheat bran 80g, corn cob 10g, soybean cake powder 10g.
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| CN109576245A (en) * | 2018-12-27 | 2019-04-05 | 北京华美源生物科技有限公司 | Barley diet barley enzyme preparation and its zymotechnique |
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