CN101067130A - Process of producing beta-mannase with Aspergillus niger - Google Patents
Process of producing beta-mannase with Aspergillus niger Download PDFInfo
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- CN101067130A CN101067130A CN 200710022406 CN200710022406A CN101067130A CN 101067130 A CN101067130 A CN 101067130A CN 200710022406 CN200710022406 CN 200710022406 CN 200710022406 A CN200710022406 A CN 200710022406A CN 101067130 A CN101067130 A CN 101067130A
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Abstract
The process of producing beta-mannase with Aspergillus niger belongs to the field of enzyme engineering technology. In the process of producing beta-mannase, corn cob, bran and other crop leftovers are used as the base material; and proper amount of konjaku powder, corn milk, inorganic salt, water, etc are added for solid fermentation with Aspergillus niger WA9024 strain. The present invention can obtain beta-mannase product possessing enzyme activity of 18912-22185 IU/g in test production or 12678-15921 IU/g in industrial production. The present invention provides also the recipe of fermenting culture medium and the fermenting conditions.
Description
Technical field
The present invention relates to a kind of is main raw material with agricultural byproducts such as wheat bran, Semen Maydis powder and coarse colza meals, utilizes aspergillus niger WA9024 bacterial strain solid state fermentation to produce the method for 'beta '-mannase, belongs to technical field of enzyme engineering.
Technical background
β-1,4-D-mannosans seminose lytic enzyme (β-1,4-D-mannan mannohydrolase, EC3.2.1.78) be the main chain internal random cutting β-1 of a class from mannosans, glucomannan, polygalactomannan and galactoglucomannan etc., the lytic enzyme of 4-D-seminose glycosidic bond, (β-mannanase), belong to the hemicellulose enzyme extensively is present in lower animal, bean, marine organisms and the microorganism to be called for short 'beta '-mannase.
Along with the development and use of the mankind, and to the continuous discovery with the manna oligosaccharide physiological function of understanding in depth of beta-mannase antinutritional factor, for the research and development of 'beta '-mannase provides wide development space to the nature hemicellulase.At present, research and the production about 'beta '-mannase is mainly derived from microorganism.That microbe-derived 'beta '-mannase has is active high, cost is low, steady sources, extraction is convenient and the distinguishing features such as action pH, temperature range and Substratspezifitaet wider than animals and plants.Therefore, be widely used in fields such as food, medicine, feed, papermaking, oil production and bio-science research.
In recent years, the research and development about 'beta '-mannase has entered a new climax.In foodstuffs industry, because the continuous discovery that the manna oligosaccharide health care is worth has had more widely 'beta '-mannase and uses in food-processing.For example, add 'beta '-mannase in soluble coffee production, the quality that can not only improve soluble coffee can also increase its health care and be worth; In the production process of garden spgarden stuff, add the viscosity that 'beta '-mannase can reduce the raw material juice, not only greatly reduce the nutrient health-care function that production energy consumption has also improved garden spgarden stuff.In the fodder industry, along with the many functions of 'beta '-mannase (as: promote growth of animal, improve the animal-feed transformation efficiency; Promote nutritive ingredient digestion, improve the animal intestinal microbial ecological; Reduce the viscosity of enteron aisle Dissolve things inside, reduce the ight soil discharging, alleviate environmental pollution; Improve the biological utilisation of trace element, etc.) discovery, it is widely used in the production of various animal-feeds with a kind of form of fodder additives.Along with the continuous discovery of function in industry-by-industry of 'beta '-mannase, its demand will constantly increase, for it further researchs and produces development and the market outlook that provide wide.
At present, at home and abroad in the bibliographical information, research about the microorganism 'beta '-mannase mainly comprises: the screening and the selection by mutation of producing 'beta '-mannase microorganism (comprising fungi, bacterium, actinomycetes etc.), produce the optimization of enzymic fermentation processing condition (comprising solid-state or liquid fermentation medium and culture condition etc.), the mensuration of the separation and purification of enzyme and character (number of components, molecular weight, iso-electric point, optimum temperature and pH stability, enzyme kinetics characteristic and the Substratspezifitaet etc. that comprise enzyme), and the clone of enzyme gene and expression etc.Entered since the nineties in 20th century, widespread use along with genetic engineering technique and protein engineering, in the increasing working set with the aspects such as research of clone, expression and the avtive spot of beta-mannase gene, but present sophisticated report is also few, and relevant 'beta '-mannase solid state fermentation industrialization producer rule is not appeared in the newspapers as yet.
Li Jianfang etc. utilize ultraviolet and nitrosoguanidine mutagenesis breeding, have obtained a strain aspergillus niger mutant strain L-76-1, and its liquid fermenting producing acidic beta-mannase activity is 320U/mL; Chai Pingping etc. have optimized the liquid fermentation condition of genus bacillus WY45, and the beta-mannase enzymic activity reaches 2800U/mL; Li Jianghua etc. utilize the ultraviolet mutagenesis breeding, have obtained a strain aspergillus niger mutant strain WA9024, and its solid state fermentation produces the beta-mannase enzymic activity and reaches 8940U/g.Heck etc. optimize the solid-state fermentation culture medium of Bacillus circulans BL53 bacterial strain, and its high yield beta-mannase enzymic activity is 560U/g; Juhasz etc. have reported the influence that different carbon sources are produced Mierocrystalline cellulose and hemicellulase to Trichodermareesei, and its high yield beta-mannase enzymic activity is 4500U/g.Though existing many both at home and abroad bibliographical informations that utilize microbial liquid or solid state fermentation to produce 'beta '-mannase, but the fermenting enzyme activity is generally lower, limited the widespread use of this enzyme, especially in Application for Field such as fodder industry and manna oligosaccharide productions.
Now, the 'beta '-mannase of using as commodity is arranged also on the market, mainly contain that U.S. ChemGen company produces with U.S. ferment and domestic magnificent fragrant enzyme etc.But, hindered the widespread adoption of 'beta '-mannase owing to cost an arm and a leg.The present invention has obtained a kind of simple, easy to operate, process for solid state fermentation that 'beta '-mannase is produced in cost is low, income is big industrialization, its 'beta '-mannase fermenting enzyme activity is higher than present domestic level, is applicable to less energy-consumption, modern zymin production cheaply.
Summary of the invention
The purpose of this invention is to provide a kind of serves as the fermentation base-material with agricultural byproducts such as wheat bran, Semen Maydis powder and coarse colza meals, utilizes aspergillus niger WA9024 bacterial strain solid state fermentation to produce the method for 'beta '-mannase.
Technical scheme of the present invention:
Solid state fermentation laboratory lab scale is produced 'beta '-mannase:
Bacterial strain: aspergillus niger (4spergillus niger) WA9024, by screening of Molecular Biology Research Lab of medical college of Southern Yangtze University and preservation.This bacterial classification rolled up for the 5th phase at " Wuxi Light Industry Univ.'s journal " the 23rd the sixth of the twelve Earthly Branches, and in September, 2004 is open.
Inclined-plane seed culture medium and culture condition: peeling potato 200g/L, sucrose 20g/L, agar 20g/L, natural pH; 121 ℃ of sterilization 20min place the inclined-plane; Cooling back inoculation WA9024 bacterial strain is put 32 ℃ and is cultivated 96h.This slant medium is used for actication of culture, preservation and test tube slant seed etc.
Fermention medium and fermentation condition: the bottled 10g base-material of 250mL triangle (wherein: wheat bran 6～8g, Semen Maydis powder 1～2g, coarse colza meal 1～2g), Rhizoma amorphophalli powder 0.3～0.5g, corn steep liquor 0.3～0.5g, (NH
4)
2SO
450～100mg, KH
2PO
430～50mg, CaCl
25～10mg, CuSO
42H
2O 0.5～1mg, KI 0.5～1mg, MnSO
4H
2O 0.5～1mg, tap water 16～18mL, pH 6.0.121 ℃ of sterilization 40min, cooling back inoculation one bacterium ear WA9024 test tube slant seed is cultivated 84～96h in 31～33 ℃, during respectively turn over bent 1 time and respectively add sterilized water 1.6～1.8mL respectively at 24～28h and 44～48h.
The preparation of triangular flask wheat bran seed: the bottled 10g base-material of 250mL triangle (being wheat bran 10.0g), (NH
4)
2SO
450mg, tap water 13mL, pH 6.0.121 ℃ of sterilization 40min, cooling back inoculation WA9024 inclined-plane seed is cultivated 96h for 32 ℃, during respectively turn over bent 1 time respectively at 24h and 44h.
The preparation of koji tray wheat bran seed: take by weighing 3.0kg base-material (being wheat bran 3.0kg), (NH
4)
2SO
415g, tap water 3.9L, pH 6.0, and mixing is in the 8 layers of gauze bag of packing into, 121 ℃ of sterilization 40min, (100cm * 50cm * 10cm), inoculate 1 triangular flask wheat bran seed cultivates 96h in 32 ℃ of bent rooms to put into koji tray after the cooling, respectively turn over bent 1 time respectively at 24h and 44h during this time, and respectively add sterilized water 390mL simultaneously.
'beta '-mannase is produced in the solid state fermentation industrialization:
Inoculum: WA9024 koji tray wheat bran seed.
Fermentative medium formula: the 1000kg base-material (wherein: wheat bran 700～800kg, Semen Maydis powder 100～150kg, coarse colza meal 100～150kg), and add Rhizoma amorphophalli powder 30～50kg, corn steep liquor 30～50kg, (NH
4)
2SO
45～10kg, KH
2PO
43～5kg, CaCl
20.5～1.0kg, CuSO
42H
2O 0.05kg, KI 0.05kg, MnSO
4H
2O 0.05kg, tap water 1800～2000kg, pH 6.0.
Solid state fermentation conditions: at 6M
3Add solid-state fermentation culture medium in the steam cooker, mixing is heated to 121 ℃ with open steam, insulated sterilizing 40min, be cooled to 38～40 ℃ after, inoculation 3kg WA9024 koji tray wheat bran seed, mixing places 30M
3In the solid-state fermenter, 32～36 ℃ of intermittent air blasts are cultivated 72～84h, during respectively turn over bent 1 time and respectively add sterilized water 180～200kg respectively at 20～24h and 36～40h.
Analytical procedure:
Beta-mannase enzyme assay: in 25mL tool plug test tube A, B, the mass concentration that each adding is disposed with pH 4.8, acetate-sodium acetate buffer is 0.5% carob bean gum solution 2.4mL, behind 50 ℃ of preheating 10min, in the A pipe, add the suitably enzyme liquid of dilution of 0.1mL, 50 ℃ of accurate response 10min; Add 2.5mL 3 ', 5 '-dinitrosalicylic acid (DNS) reagent immediately respectively, in the B pipe, add 0.1mL enzyme liquid again, boil 7min; Respectively add water 5mL after the cooling, shake up; 540nm sentences the B pipe and measures A pipe absorbancy for blank, and finds corresponding reducing sugar (in seminose) content and be converted to unit of enzyme from the seminose typical curve.The enzymic activity definition: under this condition determination, the enzyme amount that produces 1 μ mol reducing sugar with per minute is defined as 1 beta-mannase unit of enzyme activity (IU).
Reducing sugar test: adopting the DNS method, is standard with the seminose.
Moisture determination: take by weighing a certain amount of wheat bran, dry to constant weight for 105 ℃.
Beneficial effect of the present invention: the advantage that the present invention gives prominence to is to utilize cheap agricultural byproducts such as wheat bran, Semen Maydis powder and coarse colza meal as the fermentation main raw material, is a kind of production method that makes full use of the nature renewable resources.Simultaneously, bacterial strain aspergillus niger of the present invention is the microorganism of generally recognized as safe (GRAS), therefore utilizes fermentation of Aspergillus niger production zymin to have safely, reliably and not produce advantages such as toxin; Utilizing aspergillus niger (Aspergillus niger) WA9024 bacterial strain solid state fermentation to produce 'beta '-mannase, meet the natural law in the ecosystem, is a kind of environmental protection and continuable production method.The present invention utilizes aspergillus niger (Aspergillus niger) WA9024 bacterial strain, the enzymic activity that the laboratory lab scale is produced 'beta '-mannase can reach 18912～22185IU/g dry medium, has improved 2.11～1.47 times than the enzymic activity (8984IU/g dry medium) of the former solid state fermentation of WA9024 bacterial strain; The enzymic activity that 'beta '-mannase is produced in industrialization is 12678～15921IU/g dry medium.
Embodiment
Below be with agricultural byproducts such as wheat bran, Semen Maydis powder and coarse colza meal serve as the fermentation main raw material, and add an amount of Rhizoma amorphophalli powder, corn steep liquor, inorganic salt and tap water etc., utilize aspergillus niger (Aspergillus niger) WA9024 bacterial strain solid state fermentation to produce the embodiment of 'beta '-mannase, but the present invention is not limited to listed several embodiment.
Embodiment 1
Bacterial strain: aspergillus niger WA9024.
Triangular flask solid-state fermentation culture medium: the bottled 10g base-material of 250mL triangle (wherein: wheat bran 6.4g, Semen Maydis powder 1.6g, coarse colza meal 2.0g), Rhizoma amorphophalli powder 0.4g, corn steep liquor 0.5g, (NH
4)
2SO
4100mg, KH
2PO
430mg, CaCl
210mg, CuSO
42H
2O 0.5mg, KI 0.5mg, MnSO
4H
2O 0.5mg, tap water 17mL, pH 6.0.
The triangular flask solid state fermentation conditions: at 121 ℃ of sterilization 40min, cooling back inoculation one bacterium ear WA9024 inclined-plane seed leaves standstill in 32 ℃ and to cultivate 84h with substratum, during respectively turn over bent 1 time and respectively add the 1.7mL sterilized water simultaneously respectively at 24h and 44h.
The above-mentioned triangular flask test-results that repeats 5 batches shows that the average enzymic activity of 'beta '-mannase of the bent material of maturing fermentation reaches the 22185IU/g dry medium.
Embodiment 2
Bacterial strain: aspergillus niger WA9024.
Solid state fermentation equipment: 30M is produced in industrialization
3Solid-state fermenter is with temperature automatically controlled air-blast device, machinery to turn over bent device, mechanical water replanishing device and mechanical drawing mechanism etc.Tank diameter 4.5m, tank body height 2.0m, real dress 1000kg base-material (in dry material), fermention medium ulking thickness 30cm.
Industrialization production solid-state fermentation culture medium: 1000kg base-material (wherein: wheat bran 800kg, Semen Maydis powder 100kg, coarse colza meal 100kg), Rhizoma amorphophalli powder 40kg, corn steep liquor 50kg, (NH
4)
2SO
410kg, KH
2PO
43kg, CaCl
21.0kg, CuSO
42H
2O 0.05kg, KI 0.05kg, MnSO
4H
2O 0.05kg, tap water 2000kg, pH 6.0.
Solid state fermentation conditions is produced in industrialization: above-mentioned fermention medium is added 6M
3The steaming and decocting under high pressure pot, with being steam heated to 121 ℃, insulation 40min, be cooled to 38～40 ℃ after, inoculation 3kg WA9024 koji tray wheat bran seed, mixing places 30M
3In the solid-state fermenter, adopt intermittent air blast that temperature is controlled at 32 ℃, constant temperature culture 84h, during respectively turn over bent 1 time respectively at 24h and 40h, and respectively add sterilized water 200kg simultaneously.
The above-mentioned industrialization mill trial results that repeats 3 batches shows that the bent material of maturing fermentation is through 45～50 ℃ of hot-air seasonings (dry medium water content about 8%), and the average enzymic activity of 'beta '-mannase reaches the 12678IU/g dry medium.
Embodiment 3
In order to help the growth of thalline, reach the purpose that shortens fermentation period and control living contaminants, present embodiment adopts the low alternating temperature master mode in the preceding high back of solid state fermentation temperature, and the solid-state fermentation culture medium prescription is with embodiment 2.
Solid-state fermentation culture medium places 30M after sterilization, cooling, inoculation
3In the solid-state fermenter.(1) (0～20h): 0～8h is with blast inlet and air outlet Close All, and temperature controller is arranged on 36 ℃ for earlier fermentation; 8～12h opens 1/3 with blast inlet and air outlet, and 12～20h opens big blast inlet and air outlet gradually subsequently, and until whole unlatchings, temperature still is controlled at 36 ℃.(2) (20～56h): 20h turns over Qu Yici and adds sterilized water 200kg simultaneously ferment middle, and design temperature is turned down to 35 ℃; 36h turns over Qu Yici again and adds sterilized water 200kg simultaneously, temperature will be set turn down to 34 ℃; 36～56h turns down temperature gradually to 32 ℃.(3) the fermentation later stage (56～72h): always remain to the 72h fermentation ends with 32 ℃.
The above-mentioned industrialization mill trial results that repeats 3 batches shows that the bent material of maturing fermentation is through 45～50 ℃ of hot-air seasonings (dry medium water content about 8%), and the average enzymic activity of 'beta '-mannase reaches the 14028IU/g dry medium.
Embodiment 4
The ratio of wheat bran and coarse colza meal in the change solid state fermentation base-material, all the other are with embodiment 3.
1000kg base-material (wherein: wheat bran 750kg, Semen Maydis powder 100kg, coarse colza meal 150kg), and add Rhizoma amorphophalli powder 40kg, corn steep liquor 50kg, (NH
4)
2SO
410kg, KH
2PO
43kg, CaCl
21.0kg, CuSO
42H
2O 0.05kg, KI 0.05kg, MnSO
4H
2O 0.05kg, tap water 2000kg, pH 6.0.Fermention medium places 30M after sterilization, cooling, inoculation
3In the solid-state fermenter, fermentation condition is with embodiment 3.
The above-mentioned industrialization mill trial results that repeats 3 batches shows that the bent material of maturing fermentation is through 45～50 ℃ of hot-air seasonings (dry medium water content about 8%), and the average enzymic activity of 'beta '-mannase reaches the 15921IU/g dry medium.
Claims (2)
1, a kind of lab scale is produced the method for 'beta '-mannase, it is characterized in that with wheat bran, Semen Maydis powder and coarse colza meal being the base-material of solid-state fermentation culture medium, and add an amount of Rhizoma amorphophalli powder, corn steep liquor, inorganic salt and tap water, in the 250mL triangular flask, utilize aspergillus niger (Aspergillus niger) WA9024 bacterial strain solid state fermentation to produce 'beta '-mannase;
The solid-state fermentation culture medium prescription is: the bottled 10g base-material of 250mL triangle, wherein: wheat bran 6～8g, Semen Maydis powder 1～2g, coarse colza meal 1～2g, and add Rhizoma amorphophalli powder 0.3～0.5g, corn steep liquor 0.3～0.5g, (NH
4)
2SO
450～100mg, KH
2PO
430～50mg, CaCl
25～10mg, CuSO
42H
2O 0.5～1mg, KI 0.5～1mg, MnSO
4H
2O 0.5～1mg, tap water 16～18mL, pH 6.0;
Solid state fermentation conditions is: 121 ℃ of fermention mediums sterilization 40min, inoculation one bacterium ear WA9024 inclined-plane seed in cooling back is cultivated 84～96h in 31～33 ℃, during respectively turn over bent 1 time and respectively add sterilized water 1.6～1.8mL respectively at 24～28h and 44～48h.
2, the method for 'beta '-mannase is produced in a kind of industrialization, it is characterized in that with wheat bran, Semen Maydis powder and coarse colza meal being the base-material of solid-state fermentation culture medium, and adds an amount of Rhizoma amorphophalli powder, corn steep liquor, inorganic salt and tap water, at 30M
3In the solid-state fermenter, utilize the industrialization of aspergillus niger (Aspergillus niger) WA9024 bacterial strain solid state fermentation to produce 'beta '-mannase;
The solid-state fermentation culture medium prescription: the 1000kg base-material, wherein: wheat bran 700～800kg, Semen Maydis powder 100～150kg, coarse colza meal 100～150kg, and add Rhizoma amorphophalli powder 30～50kg, corn steep liquor 30～50kg, (NH
4)
2SO
45～10kg, KH
2PO
43～5kg, CaCl
20.5～1.0kg, CuSO
42H
2O 0.05kg, KI 0.05kg, MnSO
4H
2O 0.05kg, tap water 1800～2000kg, pH 6.0;
The solid state fermentation culture condition is: at 6M
3Add solid-state fermentation culture medium in the steam cooker, behind the mixing, be heated to 121 ℃ with open steam, insulated sterilizing 40min, be cooled to 38～40 ℃ after, inoculation 3kg WA9024 koji tray wheat bran seed, mixing places 30M
3In the solid-state fermenter, 32～36 ℃ of intermittent air blasts are cultivated 72～84h, during respectively turn over bent 1 time and respectively add sterilized water 180～200kg respectively at 20～24h and 36～40h.
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| CN100558888C CN100558888C (en) | 2009-11-11 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102031226A (en) * | 2009-10-06 | 2011-04-27 | 马旭光 | Aspergillus niger strain and application thereof |
| CN102586209A (en) * | 2012-03-15 | 2012-07-18 | 济南诺能生物工程有限公司 | Preparation method for liquid high temperature resistant acidic mannase |
| CN107805635A (en) * | 2017-12-26 | 2018-03-16 | 马鞍山市五谷禽业专业合作社 | A kind of method that β mannases are prepared using pomace solid state fermentation |
| CN108504581A (en) * | 2018-04-13 | 2018-09-07 | 甘肃省农业科学院植物保护研究所 | The solid culture and liquid fermentation process of short close trichoderma |
| CN110577946A (en) * | 2018-06-07 | 2019-12-17 | 青岛红樱桃生物技术有限公司 | Beta-mannase mutant with improved enzyme activity and heat resistance as well as encoding gene and application thereof |
| CN114437938A (en) * | 2022-01-14 | 2022-05-06 | 山东隆科特酶制剂有限公司 | High-yield high-temperature-resistant acidic beta-mannase strain and application thereof |
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2007
- 2007-05-17 CN CNB200710022406XA patent/CN100558888C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102031226A (en) * | 2009-10-06 | 2011-04-27 | 马旭光 | Aspergillus niger strain and application thereof |
| CN102586209A (en) * | 2012-03-15 | 2012-07-18 | 济南诺能生物工程有限公司 | Preparation method for liquid high temperature resistant acidic mannase |
| CN102586209B (en) * | 2012-03-15 | 2013-08-07 | 济南诺能生物工程有限公司 | Preparation method for liquid high temperature resistant acidic mannase |
| CN107805635A (en) * | 2017-12-26 | 2018-03-16 | 马鞍山市五谷禽业专业合作社 | A kind of method that β mannases are prepared using pomace solid state fermentation |
| CN108504581A (en) * | 2018-04-13 | 2018-09-07 | 甘肃省农业科学院植物保护研究所 | The solid culture and liquid fermentation process of short close trichoderma |
| CN108504581B (en) * | 2018-04-13 | 2021-06-15 | 甘肃省农业科学院植物保护研究所 | Solid culture and liquid fermentation process of short dense trichoderma |
| CN110577946A (en) * | 2018-06-07 | 2019-12-17 | 青岛红樱桃生物技术有限公司 | Beta-mannase mutant with improved enzyme activity and heat resistance as well as encoding gene and application thereof |
| CN110577946B (en) * | 2018-06-07 | 2021-01-26 | 青岛红樱桃生物技术有限公司 | Beta-mannase mutant with improved enzyme activity and heat resistance as well as encoding gene and application thereof |
| CN114437938A (en) * | 2022-01-14 | 2022-05-06 | 山东隆科特酶制剂有限公司 | High-yield high-temperature-resistant acidic beta-mannase strain and application thereof |
| CN114437938B (en) * | 2022-01-14 | 2023-06-06 | 山东隆科特酶制剂有限公司 | Strain for high-yield high-temperature-resistant acid beta-mannase and application thereof |
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|---|---|
| CN100558888C (en) | 2009-11-11 |
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Granted publication date: 20091111 Termination date: 20100517 |