CN102277343B - Biological composite enzyme - Google Patents
Biological composite enzyme Download PDFInfo
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- CN102277343B CN102277343B CN201110207442.XA CN201110207442A CN102277343B CN 102277343 B CN102277343 B CN 102277343B CN 201110207442 A CN201110207442 A CN 201110207442A CN 102277343 B CN102277343 B CN 102277343B
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- aspergillus awamori
- bacillus subtilis
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- 238000002360 preparation method Methods 0.000 claims abstract description 24
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- 239000003795 chemical substances by application Substances 0.000 claims description 28
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
Abstract
The invention discloses a biological composite enzyme, belongs to the field of biotechnology and particularly relates to a biological composite enzyme product. The product provided by the invention consists of product of culture of aspergillus awamori, lactobacillus plantarum preparation and bacillus subtilis preparation. Each gram of biological composite enzyme contains 4.0 to 9.5*10<8> bacillus subtilis, 4.0 to 8.5*10<7> aspergillus awamori, 150 to 200IU of protease, 200 to 250IU of beta-glucanase, 50 to 150 IU of xylanase, 100 to 120 IU of cellulose and 250 to 300 IU of amylase. When the biological composite enzyme is applied into soil, the soil looseness of an active layer can be promoted, the soil fertility can be improved, and the sound soil environment suitable for the roots of crops to grow. The conversion of the soil bio-enzyme has obvious effects in aspects of reducing agricultural production cost, reducing consumption of fertilizer, restoring ecological productivity of soil, effectively improving crop yield, and the like and can give full play to the efficacy of the organic matters of the soil.
Description
Technical field
The invention belongs to biological technical field, particularly compound bio-enzyme product.
Background technology
Compound bio-enzyme is widely used in the industries such as weaving, food-processing, medicine, fermentation, tobacco ageing, feed, improves soil but be applied to, and promotes that crop growth still belongs to the first time, and has no data report.
The reasonable compatibility of prozyme in the outer prozyme of unconventional born of the same parents that industrial microorganism is produced and born of the same parents, can effectively promote other microbial growth, and most of generation biological, metabolite is had to positive effect, simultaneously obvious to microbial enzyme activation, the imagination of exploitation fermentation industry formulation products has been proposed.
Researched and developed the compound enzymic preparation (soil organisms prozyme) for soil microorganisms simultaneously, conceptive from large fermentation, the earth can be regarded a fermentor tank, the growth of soil microorganisms as, the reasonable conversion of the soil organism, inestimable to the promoter action of plant-growth.We think and utilize the metabolic enzyme of industrial microorganism, promote and activate the endogenous enzyme activity of soil microorganisms and plant, promote microorganism and plant-growth, and soil is improved and improves crop yield and be significant.Due to the life-time service of chemical fertilizer, can damage flora structure that soil is suitable and the crumb structure of soil, so protection ecology and soil fertility have become the emphasis of current agricultural problem.Because agriculture production is used chemical fertilizer in a large number, plant continues to increase the dependency degree of chemical fertilizer, has suppressed soil conversion capability, and fertilizer effect is difficult to give full play to, and Soil structure should not improve.
Summary of the invention
The object of this invention is to provide compound bio-enzyme product.
Product of the present invention is by Aspergillus awamori culture.Plant lactobacillus agent and bacillus subtilis microbial agent composition, subtilis 4.0～9.5 × 10 in every gram of product of described compound bio-enzyme
8individual, Aspergillus awamori 4.0～8.5 × 10
7individual, proteolytic enzyme 150～200IU, beta-glucanase 200～250IU, zytase 50～150IU, cellulase 100～120IU, amylase 2 50～300IU.
Described compound bio-enzyme is made up of Aspergillus awamori culture 50-80 part, plant lactobacillus agent 5-10 part, bacillus subtilis microbial agent 20-40 part; Aforementioned proportion is mass ratio.
The concrete production method step of product of the present invention is as follows:
1, microbial inoculum is cultivated preparation:
Bacillus subtilis microbial inoculum is cultivated preparation: bacillus subtilis microbial agent preparation: cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete centrifugation of fermenting obtains wet thallus and fermented liquid; The complete fermented liquid that ferments is concentrated in vacuo to the 40-50% of original volume through low-temperature negative-pressure, obtain bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5-0.7: 1, and vehicle group becomes: CaCO
320-40 part, dextrin 10-20 part.Fluidised bed drying, 50 DEG C of drying temperatures.
The preparation method of plant lactobacillus agent: from inclined-plane switching culturing plants Bacterium lacticum, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete fermented liquid that ferments is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5-0.7: 1, and vehicle group becomes: CaCO
320-40 part, dextrin 10-20 part.Fluidised bed drying, 50 DEG C of drying temperatures.
Aspergillus awamori culture preparation: spawn culture, solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, and 26-35 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized-bed dry, pulverize dry thing;
2, microbial inoculum compound proportion is as follows:
Aspergillus awamori culture 50-80 part.Plant lactobacillus agent 5-10 part, bacillus subtilis microbial agent 20-40 part, the bacterial classification adopting is common subtilis (Bacillus subtilis subsp), plant lactobacillus (Lactobacillusplantarum) and Aspergillus awamori (Aspergillus awamori).
In the present invention, enzyme activity unit IU is defined as the enzyme amount of catalysis 1 micromole's substrate conversion in 1 minute.
In the present invention, adopt microorganism plate count method to detect viable count.
In the present invention, adopting conventional enzyme activity detection method to detect enzyme lives.
What in the present invention, microbial inoculum was composite compared with ratio of greater inequality example is: Aspergillus awamori culture 60-80 part.Plant lactobacillus agent 5-8 part, bacillus subtilis microbial agent 20-30 part;
This product using method is simple, every mu of ground usage quantity 0.3-1 kilogram, and cost is only 80-100 unit/mu, spray on seed dressing or the front earth's surface of pouring water vegetative period.
Beneficial effect
Through experimental results show that for many years, the conversion energy of soil organisms enzyme improves the soil compaction, acidifying, the saliferous problem that partially cause to fertilize because of long-term, sandy soil reunion, clay are loosened, strengthen water conservation, fertilizer-preserving ability, in soil, a large amount of biological enzymes has the ability of point phosphorus decomposing.But the inorganic fertilizer that life-time service is a large amount of and agricultural chemicals, disappear some probiotics extinction.So we are manured into soil with appropriate compound bio-enzyme, can impel the loosing soil of active layer, increase soil fertility, build a good soil environment that is suitable for crop root growth.The conversion of soil organisms enzyme, reducing agriculture production cost, reducing use, the recovery soil ecology soil fertility of chemical fertilizer and effectively improve the aspects such as crop yield and all can play significant effect, gives full play to the organic usefulness of soil.
Embodiment
Unless stated otherwise, in the present invention, technique means used is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention are only limited by claims.To those skilled in the art, do not deviating under the prerequisite of essence of the present invention and scope, the various changes that the reaction conditions in these embodiments, separation and Extraction condition are carried out or change also belong to protection scope of the present invention.
The following examples can make the present invention of those skilled in the art comprehend, but do not limit the present invention in any way.
Embodiment 1
Product compositing formula of the present invention is as follows:
Compound bio-enzyme product consists of: subtilis 8.0 × 10 in every gram of product
8individual, Aspergillus awamori 4.0 × 10
7individual, proteolytic enzyme 200IU, beta-glucanase 220IU, zytase 100IU, cellulase 11 0IU, amylase 2 70IU.
The concrete production method step of product of the present invention is as follows:
Microbial inoculum is cultivated preparation:
Bacillus subtilis microbial inoculum is cultivated preparation: bacillus subtilis microbial agent preparation: cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete centrifugation of fermenting obtains wet thallus and fermented liquid; The complete fermented liquid that ferments is concentrated in vacuo to 50% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.6: 1, and vehicle group becomes: CaCO
330 parts, 15 parts, dextrin.Fluidised bed drying, 50 DEG C of drying temperatures.
The preparation of plant lactobacillus agent: from inclined-plane switching culturing plants Bacterium lacticum, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete fermented liquid that ferments is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.6: 1, and vehicle group becomes: CaCO
330 parts, 16 parts, dextrin.Fluidised bed drying, 50 DEG C of drying temperatures.
Aspergillus awamori culture preparation: spawn culture, solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, and 30 DEG C are cultured to mycelia and cover with culture material, and 45 DEG C of low temperature fluidized-beds dry, pulverize dry thing;
The composite part by weight of microbial inoculum is as follows: 70 parts of Aspergillus awamori cultures, 6 parts of plant lactobacillus agent, 30 parts of bacillus subtilis microbial agents, and the bacterial classification of employing is as follows:
Subtilis (Bacillus subtilis subsp) CGMCC1.1630
Plant lactobacillus (Lactobacillus plantarum) CGMCC1.124
Aspergillus awamori (Aspergillus awamori) CGMCC3.6484
The depositary institution of above-mentioned four kinds of bacterial classifications is Chinese common micro-organisms culture presevation administrative centers.Address: No. 13, Zhongguancun N 1st Lane, Beijing City institute of microbiology of the Chinese Academy of Sciences; Postcode: 100080.
The preparation method of Aspergillus awamori microbial inoculum:
Technical scheme is as follows:
Slant strains activation culture: Aspergillus awamori slant strains is transferred on slant medium, cultivates 3 days for 27 DEG C.
Solid first order seed is cultivated: picking Aspergillus awamori slant strains accesses in 500 milliliters of triangular flasks that 100 grams of substratum are housed carries out seed culture, cultivates 3 days for 30 DEG C.
Solid secondary seed is cultivated: above-mentioned cultured solid first order seed is stirred as adding after fragment and in 5000 milliliters of triangular flasks that 1000 grams of substratum are housed, carries out seed culture, culture condition: cultivate 3 days for 30 DEG C.
Solid fermentation is cultivated: secondary shake-flask seed is pulverized, added to be equipped with in the fermentation vat of sterilising medium or pallet to mix rear cultivation, bent material culture temperature is controlled at 26-35 DEG C, humidity 80-90%, every 10 hours stirrings once, incubation time 5-7 days; The cultivation of solid koji material adopts conventional bent material culture technique; Treat that culture material covers with mycelia and can finish to cultivate, substratum is in advance through high temperature steaming sterilising treatment, 121 DEG C of sterilising conditions control temperature, 1 hour time.
Drying and crushing: fermentation ends culture material is dried on fluidized-bed or other drying plants, and drying temperature is controlled at 60 DEG C, is dried to moisture content below 10%, then solid culture medium is pulverized, and crushing material aperture is more than 60 orders.
Substratum composition: solid material: wheat bran 80%, soybean cake powder 10%, W-Gum 10%, adds equivalent tap water; Initial pH nature.
The preparation method of subtilis:
1. the acquisition of fermented liquid: adopt slant strains to spread cultivation step by step and obtain fermentation of bacillus subtilis liquid;
(1) first order seed is cultivated: subtilis slant strains is accessed in 500 ml shake flasks to 100 milliliters of substratum loading amounts, 180 revs/min of rotary shaking tables, 30 DEG C of culture temperature, incubation time 24 hours;
(2) secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum size access of 10%, culture condition is identical with first order seed;
(3) three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed shaking flasks to 1000 milliliters of substratum loading amounts, 100 revs/min of rotary shaking tables, 30 DEG C of culture temperature, incubation time 24 hours with 10% inoculum size;
(4) first class seed pot is cultivated: the first class seed pot by three grades of seeds taking 10% inoculum size access cubic capacity as 150L, fermention medium loading amount 100L, 28 DEG C of culture temperature, 100 revs/min of stirring velocitys, ventilation (V/V) 1: 0.5, tank pressure 0.05Mpa, incubation time 24 hours;
(5) fermentation culture: first class seed pot bacterial classification is accessed to cubic capacity as 1.5 tons of secondary seed tanks taking 10% inoculum size, 1 ton of fermention medium loading amount, 28 DEG C of culture condition culture temperature, 100 revs/min of stirring velocitys, ventilation (V/V) 1: 0.5, tank pressure 0.05Mpa, incubation time 24 hours.
Substratum composition: glucose 6%, yeast extract 1%, peptone 0.2%, CaC031%, pH6.8.
The preparation method of plant lactobacillus agent:
(1) first order seed is cultivated: plant lactobacillus bacterial classification is accessed in 500 ml shake flasks to 100 milliliters of substratum loading amounts, 30 DEG C of culture temperature, incubation time 24 hours;
(2) secondary seed is cultivated: by first order seed, according in 500 milliliters of secondary seed shaking flasks of inoculum size access of 10%, culture condition is identical with first order seed;
(3) three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed shaking flasks to 1000 milliliters of substratum loading amounts, 30 DEG C of culture temperature, incubation time 24 hours with 10% inoculum size;
(4) first class seed pot is cultivated: the first class seed pot by three grades of seeds taking 5% inoculum size access cubic capacity as 150L, fermention medium loading amount 100L, 30 DEG C of culture temperature, tank pressure 0.05Mpa, incubation time 18 hours;
(5) fermentor cultivation: first class seed pot bacterial classification is accessed to cubic capacity as 3 tons of secondary seed tanks taking 5% inoculum size, 2 tons of fermention medium loading amounts, 30 DEG C of culture condition culture temperature, tank pressure 0.05Mpa, incubation time 22 hours.
Substratum consists of: casein peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 0.5%, sodium acetate 0.5%, citric acid diamines 0.2%, Tween 80 0.1%, K2HPO4 0.2%, MgSO4.7H20 0.02%, MnSO4.H2O 0.005%, CaCO32%, agar 1.5%, pH6.8.
Embodiment 2
Substantially with embodiment 1
Product compositing formula of the present invention is as follows:
Compound bio-enzyme product consists of: subtilis 4.0 × 10 in every gram of product
8individual, Aspergillus awamori 4.0 × 10
7individual, proteolytic enzyme 150IU, beta-glucanase 250IU, zytase 50IU, cellulase 100IU, amylase 2 50IU.
The concrete production method step of product of the present invention is as follows:
Microbial inoculum is cultivated preparation:
Bacillus subtilis microbial inoculum is cultivated preparation: bacillus subtilis microbial agent preparation: cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete centrifugation of fermenting obtains wet thallus and fermented liquid; The complete fermented liquid that ferments is concentrated in vacuo to 50% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5: 1, and vehicle group becomes: CaCO
320 parts, 10 parts, dextrin.Fluidised bed drying, 50 DEG C of drying temperatures.
The preparation method of plant lactobacillus agent: from inclined-plane switching culturing plants Bacterium lacticum, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete fermented liquid that ferments is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution.Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.7: 1, and vehicle group becomes: CaCO
340 parts, 20 parts, dextrin.Fluidised bed drying, 50 DEG C of drying temperatures.
Aspergillus awamori culture preparation: spawn culture, solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, and 30 DEG C are cultured to mycelia and cover with culture material, and 45 DEG C of low temperature fluidized-beds dry, pulverize dry thing;
The composite part by weight of microbial inoculum is as follows: 50 parts of Aspergillus awamori cultures, 10 parts of plant lactobacillus agent, 40 parts of bacillus subtilis microbial agents.
The experiment of product effect
Selection experimental field and test design: tested 27 days-September 30 April in 2009 and carry out in Ba Bao village, dapple pond town, Yanchi county Ningxia.
Experimental plot reaches 10 mu of field maize plantings, uses 0.5 kilogram every mu of product of the present invention respectively in the time of plantation, emerges and uses 0.5 kilogram of invention product by loose ground mode about 1 month, and control group uses conventional fertilizer.
Invention product uses corn field corn yield to reach 650 kilograms, and control group reaches 500 kilograms; The use of invention product makes corn per mu yield improve 30%.This plot was at the 2nd year plant spring wheat, and spring wheat production has reached 400 kilograms, has improved 20% than control group per unit area yield.And experimental plot Soil structure is good, without bulk and hardening.
Claims (4)
1. a compound bio-enzyme, is characterized in that described compound bio-enzyme is made up of Aspergillus awamori culture, plant lactobacillus agent and bacillus subtilis microbial agent, subtilis 4.0～9.5 × 10 in every gram of product of described compound bio-enzyme
8individual, Aspergillus awamori 4.0～8.5 × 10
7individual, proteolytic enzyme 150～200IU, beta-glucanase 200～250IU, zytase 50～150IU, cellulase 100～120IU, amylase 2 50～300IU; Described compound bio-enzyme is made up of Aspergillus awamori culture 50-80 part, plant lactobacillus agent 5-10 part, bacillus subtilis microbial agent 20-40 part;
Bacillus subtilis microbial inoculum is cultivated preparation: cultivate subtilis from inclined-plane switching, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete centrifugation of fermenting obtains wet thallus and fermented liquid; The complete fermented liquid that ferments is concentrated in vacuo to the 40-50% of original volume through low-temperature negative-pressure, obtain bacterium concentrated solution; Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5-0.7:1, and vehicle group becomes: CaCO
320-40 part, dextrin 10-20 part; Fluidised bed drying, 50 DEG C of drying temperatures;
The preparation method of plant lactobacillus agent: from inclined-plane switching culturing plants Bacterium lacticum, the seed liquor after spreading cultivation is step by step transferred in fermentor tank, and the complete fermented liquid that ferments is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains bacterium concentrated solution; Add carrier: in concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5-0.7:1, and vehicle group becomes: CaCO
320-40 part, dextrin 10-20 part; Fluidised bed drying, 50 DEG C of drying temperatures;
Aspergillus awamori culture preparation: spawn culture, solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, and 26-35 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized-bed dry, pulverize dry thing.
2. compound bio-enzyme as claimed in claim 1, is characterized in that described Aspergillus awamori culture 60-80 part, plant lactobacillus agent 5-8 part, bacillus subtilis microbial agent 20-30 part;
3. compound bio-enzyme as claimed in claim 1, is characterized in that described compound bio-enzyme consists of: subtilis 4.0 × 10 in every gram of product
8individual, Aspergillus awamori 4.0 × 10
7individual, proteolytic enzyme 150IU, beta-glucanase 250IU, zytase 50IU, cellulase 100IU, amylase 2 50IU.
4. compound bio-enzyme as claimed in claim 1, is characterized in that 50 parts of described Aspergillus awamori cultures, 10 parts of plant lactobacillus agent, 40 parts of bacillus subtilis microbial agents.
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| CN102618525B (en) * | 2012-04-16 | 2013-09-11 | 苏州昆蓝生物科技有限公司 | Compound enzyme preparation for cotton planting |
| CN102888356B (en) * | 2012-09-03 | 2014-04-02 | 新疆农业科学院土壤肥料与农业节水研究所 | Bacillus subtilis, microbial fertilizer prepared by using bacillus subtilis, method for preparing microbial fertilizer by using bacillus subtilis, and application of microbial fertilizer |
| CN103626531B (en) * | 2013-11-20 | 2015-06-17 | 邵素英 | Compound fertilizer and preparation method thereof |
| CN103739322B (en) * | 2013-12-30 | 2016-02-10 | 邵素英 | A kind of preparation method of composite bio-fertilizer |
| CN103773717B (en) * | 2013-12-30 | 2016-04-20 | 中北大学 | A kind of high-effective microorganism is fertile |
| CN108753639A (en) * | 2014-01-23 | 2018-11-06 | 天津工业大学 | A kind of preparation method of efficient ecological compoiste fertilizer |
| CN104341239A (en) * | 2014-09-22 | 2015-02-11 | 北京科慧通智慧科技有限公司 | Biofertilizer |
| CN104761373A (en) * | 2015-03-31 | 2015-07-08 | 安徽中农化工国际贸易有限公司 | Organic bio-bacterial manure and preparation method thereof |
| CN104788219A (en) * | 2015-04-24 | 2015-07-22 | 苏州市新泾村农业基地专业合作社 | Biological enzyme organic compound fertilizer for tomatoes |
| CN104803791A (en) * | 2015-04-24 | 2015-07-29 | 苏州市新泾村农业基地专业合作社 | Biological enzyme organic compound fertilizer for pepper |
| CN104892187A (en) * | 2015-06-10 | 2015-09-09 | 苏州市小林农业科技发展有限公司 | Biological enzyme organic compound fertilizer for citrus maxima |
| CN106753396A (en) * | 2016-11-29 | 2017-05-31 | 彭光简 | A kind of micro-organism enzyme preparation of organic farm soils improvement |
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| CN100364932C (en) * | 2005-07-29 | 2008-01-30 | 殷汝新 | Foliage bacterial fertilizer, its preparation method and use |
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