CN1733896A - Bacteria for improving agricultural production environment and the quality of products and yield, and its preparation method - Google Patents
Bacteria for improving agricultural production environment and the quality of products and yield, and its preparation method Download PDFInfo
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Abstract
The invention provides a biological bacterium for improving environment of agricultural production and the quality of agricultural products, and its preparing process, whrein three kinds of beneficial bacteria including Lactobacillus bulgaricus, Saccharomyces cerevisiare and Rhodospirillum rabrum are employed for culture by using aerobic processes and anaerobic process separately or in combination, the bacterium can integrate effective bacterium with its metabolic products, the effective bacteria can be symbiotic and form dominant bacteria group in the rhizosphere microflora of the soil, thsu changing the soil into a reduced state.
Description
Technical field the invention belongs to the agriculture microbial preparation technical field of plantation, specifically a kind of microbial inoculum that improves agriculture production environment, raising agricultural product quality and output and preparation method thereof.
Background technology causes the usually unconventional morbid state growth of farm crop and livestock and poultry owing to abusing chemical fertilizer, agricultural chemicals, fodder additives, oxytocic etc., and induces a series of problems such as environmental pollution, ecological degeneration, and pollution-free industry has caused the concern of global range.
Pursue the green theme that has become current era.Produce pollution-free, nuisanceless, the eutrophic organic or green food of safety become improve environment, improve human foods security and trophicity, the effective measure of enhancement HUMAN HEALTH.A series of related measures have successively been taked at aspects such as producing, processing and selling and market cultivations in countries in the world.Passed through " the food quality protection method " of this state in 1996 as the U.S., making of agricultural chemicals has been used as strict restriction, the special requirement infant foods can not contain any chemical pesticide residue.The turn of the century, held " Pesticide Action Network " international conference at stockholm, capital of sweden, its theme is " agricultural and a food safety ", conference appeals to reduce the use of agricultural chemicals as far as possible, to guarantee the security of agricultural-food, walks the road of Sustainable development.In recent years, the U.S., Europe, Japan have issued a series of Organic food laws, rules in succession, organize clearly formulated " Organic farming standard " by the international Organic farming that more than 90 more than 600 tame member unit of country form, basic one is exactly not use artificial synthetic fertilizer, agricultural chemicals, growth regulator and fodder additives; " European Union's Organic food method " regulation does not have to build the Organic food guarantee system that conforms to European Union, will be put into the 3rd state, breaks off its outlet approach.Along with the adding of WTO, global economic integration progress is further accelerated, and China's economic especially rural economy will be subjected to unprecedented challenge---and Chinese agricultural and animal products quality descends to degree very urgent, that need to be resolved hurrily.From long-term interest, market will be opened, and Trade Structure and quantity all can change, and organic or green food can capture market for farm products very soon, and this will make the green product of China be faced with formidable challenges and test, and situation is very urgent.
Reproduce beautiful mountains and rivers, recover natural, ecological, become human be self Sustainable Production and the strong desire of reinventing good life.Bread is the staff of life, and along with improving constantly of people's living standard, human consumption's idea of 21st century no longer is quantitative satisfied, but the raising of quality, the lifting of taste.Green food no longer is the topic of people's sitting around gossiping after dinner, and real has entered huge numbers of families, becomes the staple food on the dining table.People need green food, and this will create irreplaceable selling market for the development of pollution-free industry.The effect of microorganism in the ecosystem is irreplaceable, and utilizing effective microorganism preparation to improve envrionment conditions is main contents during current sustainability is produced.
At present, related microorganism preparation or bacterial manure emerge in an endless stream just like emerging rapidly in large numbersBamboo shoots after a spring rain, and have fully shown the space of prominence of beneficial microorganism in the ecological agriculture is produced.So far, microbial preparation or fertilizer that drop into to use have many kinds: aspect fixedly nitrogen, increase soil fertility, have " the wheat treasured " in Shaanxi, the wheat in Hebei to want border association nitrogen fixation microbial inoculum---and " fertile standing grain spirit " etc.; Transform, increase crop at material and absorb aspect the available nutrient, phosphorus decomposing, potassium decomposing bacterial manure, bioactive P-K composite fertilizer, " Tianlibao ", sky, the Qinghai company's " nine bacterium efficient composite biological fertilizers " etc. of speeding is arranged; Aspect assistance crop absorption nutrient, VA bush mycorrhiza agent, the short multiple microbial inoculum etc. of giving birth to of rhizosphere are arranged; Producing aspects such as growth regulatory substance, promotion crop growth, " 5406 " bacterial manure etc. is arranged; Producing microbiotic, preventing and treating crop pest, improving aspect the soil physical and chemical property, " 301 " starter, EM technology of " high-efficiency multi-function bio-feritlizer ", the development of Institute for Atomic Research, national academy of agricultural sciences of dragon technology company limited of Wuhan section NEW TYPE OF COMPOSITE bacterial manure, Peking University etc. are being arranged.Yet with regard to liquid bacterial agent, present applied product exists mostly that bacterial classification mixes, the bacterium number and chemical ingredients is unclear, so that phenomenon such as the mechanism of action is not clear.Cause operational difficulty for thus the examination and test of products, commercial production, quality surveillance.
Summary of the invention the object of the present invention is to provide a kind of bacterial classification and chemical ingredients is clear, the mechanism of action is clear and definite, technology is workable plantation agricultural with microbial inoculum and preparation method thereof, be intended to overcome above-mentioned deficiency, and focus on the effect, thereby particularly improve agriculture production environment and improve the quality of agricultural product aspect, so that for producing green food so that Organic food provides raw materials for production.
Above-mentioned purpose is achieved through the following technical measures:
A kind of agriculture production environment that improves, the microbial inoculum of raising agricultural product quality and output (hereinafter to be referred as: the AM microbial inoculum), effective bacterial classification of selecting for use is lactobacillus bulgaricus (Lactobacillus bulgaricus) and cereuisiae fermentum (Saccharomyces cerevisiare) and rhodospirillum rubrum (Rhodospirillum rabrum), the above two are provided by the Ministry of Agriculture microbial strains preservation administrative center, and (sample number into spectrum is respectively: IFFI 6047, ACCC 2038), the back is got a kind of the separation from soil according to routine techniques by the applicant, with three kinds of selected effective mycorhiza according to its physiological property, correspondingly take separately and mix, the method that aerobic combines with anaerobism is scientifically cultivated, obtain thus a kind of have imitate bacterial classification and its meta-bolites is the fermentation preparation of one, the contained effective total viable count of microbial inoculum 〉=1 * 10
8Individual/ml.
The characteristic of three kinds of bacterial classifications is respectively in the AM microbial inoculum provided by the invention:
Lactobacillus bulgaricus (Lactobacillus bulgaricus): cell is shaft-like, form is various, from long, thin, crooked, that clavate is shaft-like having to rod-short, tangible chaining does not move usually yet, and the sporter then has peritrichous, the anodontia spore, Gram-positive, little aerobic, on solid medium, cultivate, press and be filled with 5-10%CO at anaerobic condition or minimizing oxygen usually
2Can increase the surface growth thing under the situation, acidproof, optimal pH 5.5-6.2 generally can grow under pH5 or lower situation, neutral or just can reduce growth velocity usually under the alkaline condition.The fermentation using lactobacillus bulgaricus type is a homofermentation, and institute's lactic acid producing does not have the optics activity, growth temperature 30-40 ℃, have stronger acid producing ability, and breeding, metabolism power is strong, can promptly occupy growing environment.
Cereuisiae fermentum (Saccharomyces cerevisiare): in wort, grow: 28 ℃ after three days, the cell circle, inferior circular, avette, oval or cylindrical arrive microscler.Single, Cheng Shuan becomes short chain or tuftlet once in a while.Width and size by cell are divided into three groups, and intermediary or transitional type are in various degree also arranged between three groups: first group of cell is big, micron, and second group of cell is little, micron, the 3rd group be middle monoid, micron, can run into and reach 30 microns or longer filiform cell.Form precipitation, faint or incomplete ring are arranged once in a while.After the room temperature one month, precipitation is arranged, Chang Youhuan has island or moistening mycoderm once in a while.Growing on the wort agar: 28 ℃ after three days, the cell circle, inferior circular, avette, oval or cylindrical arrive microscler.Single, Cheng Shuan becomes short chain or tuftlet once in a while.Width and size by cell are divided into three groups, intermediate type is also arranged between three groups, or transitional type in various degree: first group of cell is big, 3.5-10.0 * 5.0-14.5-19 micron, second group of cell is little, 2.5-7.0 * 4.5-10.5-19 micron, and the 3rd group is intermediate type, 3.0-8.0 * 4.0-11.5-18.0 micron can run into and reaches 30 microns or longer filiform cell.The streak culture butteriness, seldom crisp, cream-colored to shallow milk brown.Usually dimpling is quite smooth once in a while, and level and smooth, cushion has wrinkle or ripple shape to pleat is arranged, and the face of excipuliform, or nick once in a while is little once in a while fan-shaped, particularly near edge.Light is to dark slightly.The edge is wavy, splintery or annular saw profile of tooth once in a while, and irregular once in a while teeth shape, or there is pseudohypha at the edge.After the room temperature one month, the shallow milk grey of culture, shallow milk brown are to light brown.Projection is arranged, and topical type is to smooth slightly, and little expansion smooths to cushion, and streak shape, fan-shaped, concave surface have young bird once in a while slightly, or the ripple shape is shinny to dark slightly to wrinkle is arranged.Full edge, wavy, annular saw profile of tooth are to splintery, and often there is pseudohypha at the edge.Pseudohypha: cultivate on the Corn Meal Agar with cover plate, pseudohypha archetype has branch once in a while.Thecasporous formation: vegetative cell directly is transformed into ascus usually, each ascus 1-4 circular thecaspore to flat ellipse.Application feature: cereuisiae fermentum except beer brewing, alcohol and other beverages, the system bread of also can leavening dough.The VITAMIN of thalline, protein content height, can do edible, medicinal and fodder yeast, can extract nucleic acid, ergosterol, glutathione, cytochrome C, thromboplastinum, coenzyme A, Triphosaden, Ceroline (a kind of fat that cereuisiae fermentum is contained) again, its saccharase can be used for Nulomoline and chocolate manufacturing.In some steroid conversion of compounds, can replace a part of chemical reaction.Cereuisiae fermentum commonly used is measured vitamin H, pantothenic acid, VitB1, is given a tongue-lashing the alcohol of trembling, inositol etc. in the microbioassay of VITAMIN.
Rhodospirillum rubrum (Rhotlospirillum rabrum): this Pseudomonas photosynthetic bacteria group, its cellular form are arc, 0.8-1.0 μ m, spirrillum, long 7-10 μ m, diameter 1.5-2.5 μ m, under unaccommodated situation, Gram-negative different shape and big or small abnormal cell can appear,, during the anaerobism liquid culture, initial pinkiness, after present intense violet color, under illumination, absolute anaerobic growth is based on photosynthetic metabolite, and is facultative aerobic, having under light anaerobism or the dark little aerobic condition and can both grow.Usually garnet is red especially on the ethanol substratum; Contain haematochrome, have the ability that absorbs the 550nm light wave; Except that simple organism, adding a small amount of yeast extract paste is necessary to growth, pH6-8.5, optimum temperuture 30-35 ℃.
The above-mentioned preparation method who improves agriculture production environment, improves the AM microbial inoculum of agricultural product quality and output may further comprise the steps:
A kind of described preparation method who improves agriculture production environment, improves the microbial inoculum of agricultural product quality and output is characterized in that it may further comprise the steps:
(1) the female kind in test tube slant prepares
When preparation lactobacillus bulgaricus test tube slant mother plants, adopt with glucose, CaCO
3And soybean sprout juice is main substratum; When preparation cereuisiae fermentum test tube slant mother plants, adopt the substratum based on glucose, peptone, yeast extract paste and maltose, the female kind in the test tube slant of the two all prepares by ordinary method
When preparation rhodospirillum rubrum test tube slant mother planted, its substratum adopted with NaCl, CH
3COONa, NH
4Cl, NaHCO
3, K
2HPO
4, MgSO
4.7H
2O and somatomedin liquid and liquid microelement are main prescription, take the method illumination cultivation with the whiteruss sealing
(2) single seed liquor preparation
(1) preparation of lactobacillus bulgaricus seed liquor
During the preparation primary seed solution, its nutrient solution is selected for use with glucose, yeast extract paste, extractum carnis, peptone, CaCO
3And inorganic salt are main prescription, or select for use with glucose, peptone, extractum carnis, yeast extract paste or yeast powder, sucrose, lactose, CH
2COONa, dibasic ammonium citrate, tween 80 and inorganic salt are main prescription, conventional aseptic technique inoculation, and shaking culture is treated viable count in the nutrient solution 〉=1 * 10
8Individual/ml, pH3.8-5.4 get final product during tool yogurt milk fragrance simultaneously
The preparation of secondary seed solution: nutrient solution adopts the prescription based on glucose, soya bean/big bean sprouts juice, boil sterilization, conventional aseptic technique, insert primary seed solution 5%, aerated culture at first, seal anaerobism then and cultivate, during constantly put the flatulence of fermenting, treat to get final product when nutrient solution is similar to primary seed solution
If need three grades of seed liquor, except that bacterial classification is that secondary seed solution, inoculum size increase to the 7-10%, other is with the preparation of secondary seed solution
(2) preparation of cereuisiae fermentum seed liquor
The preparation of primary seed solution: nutrient solution is selected the prescription based on new sweet wort Bris 12 or glucose and soybean sprout juice for use, conventional aseptic technique, and inoculation, shaking culture is treated viable count 〉=1 * 10 in the nutrient solution
7Individual/ml, pH4-5.8 get final product during the tool wine flavour simultaneously
Two---the preparation of three grades of seed liquor: copy above-mentioned first order seed liquid and preparation method thereof to amplify, difference is to boil sterilization, connects one-level or previous stage seed liquor about 10%, aerated culture
(3) preparation of rhodospirillum rubrum seed liquor
The preparation of primary seed solution: nutrient solution is selected for use with CH
3COONa, peptone, NH
4Cl, NaCl and inorganic salt, somatomedin liquid and liquid microelement are main prescription, in the Glass Containers of packing into, and conventional aseptic technique, inoculation, leave standstill, incandescent light shines on four sides, and closing anaerobic is cultivated, and treats that nutrient solution changes scarlet, bacterium viable count 〉=1 * 10 of cultivating into by original khaki color
9Individual/ml, pH is near neutral, and gets final product when having strong rotten-egg odour
The preparation of secondary seed solution: nutrient solution is selected for use with CH
3COONa, peptone, soybean sprout juice, NaCl, NH
4Cl and some other inorganic salt are main prescription, boil sterilization, and conventional aseptic technique inoculation inserts primary seed solution 5%, copies the primary seed solution cultural method to cultivate, and treats to get final product when nutrient solution is similar to primary seed solution
If need three grades of seed liquor of preparation, can imitate the secondary seed solution preparation, only inoculum size can increase to 10-15%
(3) compound seed liquid preparation
Substratum adopts based on oral glucose, the molasses of Bris more than 80 and the prescription of soybean sprout juice; Or adopt to add the prescription of glucose and fresh milk in the soybean sprout juice, boil sterilization, insert lactobacillus bulgaricus secondary or three grades of seed liquor 10%, cereuisiae fermentum secondary or three grades of seed liquor 5% respectively, aerated culture is treated effective bacterium viable count 〉=1 * 10 in the nutrient solution
9Individual/ml, pH4-4.5, and get final product during the sour-sweet wine flavour of tool
(4) preparation of AM microbial inoculum product or production
Nutrient solution is made up of following ingredients: soybean sprout filtered juice 15-30%, Bris 80 above molasses 5%, glucose 1.5-2%, urea 0.25%, and CaCO
3, rare earth, N.F,USP MANNITOL, fresh milk, several micro-inorganic salt have also been added in addition, boil sterilization, the plastics mash-back of packing into while hot, trickle is cooled to 35 ℃, inserts compound seed liquid 15-20%, EM stoste 5% same day respectively, cultivates under 30-35 ℃ of room temperature and aerobic conditions, after about 3-4 days, insert rhodospirillum rubrum two or three grades of seed liquor 10%, tighten the fermentation bung at once, sealing, carrying out anaerobism cultivates, left standstill under 30-35 ℃ 8-12 days, during constantly loosening fermentation bung so that emit fermentation flatulence, aerogenesis not to end, so far, product A M microbial inoculum is made.
Above-mentioned bacterial preparation process specifically may further comprise the steps:
The female kind in 1 test tube slant prepares
1.1 female kind the in lactobacillus bulgaricus test tube slant prepares
(1) culture medium prescription: glucose 40g, CaCO
320g milk 2ml, agar 18-20g boils and gets 10% soybean sprout juice and be settled to 1000ml, is sub-packed in the test tube
(2) sterilization, inoculation and cultivate: conventional autoclaving, 30-33 ℃ of cultivation of inoculation 5-7 days smeared in pendulum inclined-plane aseptic technique, covers with the inclined-plane to lawn
1.2 female kind the in cereuisiae fermentum test tube slant prepares
(1) culture medium prescription: maltose 1.5g, yeast extract paste 3g; Peptone 5g, glucose 10g, agar 15-18g, water adds to 1000ml, is sub-packed in the test tube
(2) sterilization, inoculation and cultivate: similar to the female preparation method of kind of above-mentioned Bulgaria, but 28-32 ℃ of cultivation 2-3 days treated to be covered with on the inclined-plane white lawn and got final product
1.3 female kind the in rhodospirillum rubrum test tube slant prepares
(1) culture medium prescription: NaCl 0.5-2g, NH
4Cl 1g, NaHCO
31g (filtration sterilization), K
2HPO
40.2g, MgSO
47H
2O 0.2g, CH
3COONa 1-5g, somatomedin liquid
(a)1ml (filtration sterilization, nutrient solution sterilization back adds), liquid microelement
(b)1ml, agar 15-18g adds water to 1000ml, and the pH nature is sub-packed in the test tube
(a) somatomedin liquid: vitamins B
110mg, Buddhist nun restrain butyric acid 1mg, para-amino benzoic acid 1mg, and vitamin H (IAA) 0.01mg, water is settled to 1000ml
(b) liquid microelement: FeCl
350mg, CuSO
40.5mg, H
3PO
410ml, NaCl 20.5mg, ZnSO
410mg, CO (NO
3)
25mg, water is settled to 1000ml
(2) sterilization, inoculation: prepare similarly to female kind of above-mentioned lactobacillus bulgaricus, but with the whiteruss sealing, 30-35 ℃, illumination (5000-10000lx) was cultivated 14-50 days, treated to occur on the inclined-plane red lawn and got final product
2 single seed liquor preparations
2.1 the preparation of lactobacillus bulgaricus seed liquor
2.1.1 the preparation of primary seed solution
(1) nutrient solution prescription
1. peptone 2g, extractum carnis 3g, yeast extract paste 3.5g, glucose 40g, K
2HPO
40.5g, KH
2PO
40.5g, MgSO
47H
2O 0.5g, CaCO
32g, vitamin complex 0.01g (1) adds water to 1000ml, pH6.8
2. peptone 10g, dibasic ammonium citrate 2g, yeast extract paste or yeast powder 5g, extractum carnis 10g, MgSO
47H
2O 0.2g, MnSO
40.05g, glucose 20g, sucrose 5g, lactose 5g, CH
2COONa5g, K
2HPO
42g, tween 80 1ml, water adds to 1000ml, and pH6.4 sterilized 15 minutes down for 121 ℃
(2) sterilization and inoculation: conventional autoclaving, aseptic technique inoculation when being cooled to 35 ℃, every 1000ml nutrient solution connects test tube, and (18 * 180mm) inclined-planes are female plants 1
(3) cultivate: under the 35-38 ℃ of condition, 180 rev/mins shaking culture 2-3 days
(4) index: bacterium viable count 〉=1 * 10 of cultivating
8Individual/ml, pH3.8-5.4, tool yogurt milk fragrance
2.1.2 the preparation of secondary seed solution
(1) nutrient solution prescription: glucose 50g, vitamin complex 50mg (50ppm), 10% soya bean (soybean) bud boiled about 30 minutes, got its juice and added to 1000ml, the pH nature
(2) sterilization inoculation: boil sterilization in 5-10 minute, insert primary seed solution 5% when being cooled to 35 ℃
(3) cultivate: 35-40 ℃, aerated culture 2-3 days, seal anaerobism then and cultivated 2-3 days, during constantly pine lid venting
(4) index: bacterium viable count 〉=1 * 10 of cultivating
9Individual/ml, other index is with the one-level seed liquor
If need three grades of seed liquor, except that bacterial classification is that secondary seed solution, inoculum size increase to the 7-10%, other is with the preparation of secondary seed solution
2.2 the preparation of cereuisiae fermentum seed liquor
2.2.1 the preparation of primary seed solution
(1) nutrient solution prescription
1. new sweet wort (Bris 12), pH5-6.4
2. soybean sprout 100g (boiling about 30 minutes) gets its juice constant volume 1000ml, wherein adds glucose 50g, pH6-7
(2) disinfection inoculation: conventional autoclaving, aseptic technique when being cooled to 30 ℃, every 1000ml nutrient solution inserts test tube, and (18 * 180mm) inclined-planes are female plants 1
(3) cultivate 28-32 ℃, 180-200 rev/min shaking culture 1-3 days
(4) index: bacterium viable count 〉=1 * 10 of cultivating
7Individual/ml, pH4-5.8, tool wine flavour
2.2.2 two---the preparation of three grades of seed liquor
Copy above-mentioned first order seed liquid and preparation method thereof to amplify, difference is to boil sterilization, connects one-level or previous stage seed liquor about 10%, aerated culture
2.3 the preparation of rhodospirillum rubrum seed liquor
2.3.1 the preparation of primary seed solution
(1) nutrient solution prescription: peptone 2g, NH
4Cl 1g, K
2HPO
40.2g, CH
3COONa 3g, MgSO
47H
2O 0.2g, NaCl 1g, somatomedin liquid
(a)1ml (filtration sterilization, nutrient solution sterilization back adds), liquid microelement
(b)1ml adds water to 1000ml, and the pH nature is in the Glass Containers of packing into
(a) somatomedin liquid: vitamins B
110mg, Buddhist nun restrain butyric acid 1mg, para-amino benzoic acid 1mg, and vitamin H (IAA) 0.01mg, water is settled to 1000ml
(b) liquid microelement: FeCl
350mg, CuSO
40.5mg, H
3PO
410ml, NaCl 20.5mg, ZnSO
410mg, CO (NO
3)
25mg, water is settled to 1000ml
(2) disinfection inoculation: conventional autoclaving, be cooled to about 35 ℃ after, every 1000ml inserts test tube, and (18 * 180mm) inclined-planes are female plants 2
(3) cultivate: airtight, leave standstill, anaerobism is cultivated, and the 40-60W incandescent light shines on four sides.Incandescent light 36-38 ℃, was cultivated 10-15 days apart from glass culturing bottle 10-15cm
(4) index: nutrient solution changes scarlet into by khaki color, bacterium viable count 〉=1 * 10 of cultivating
9Individual/ml, pH has strong rotten-egg odour near neutral
2.3.2 the preparation of secondary seed solution
(1) nutrient solution prescription: NaCl 1g, NH
4Cl 1g, K
2HPO
40.2g, MgSO
47H
2O 0.2g, peptone 1.5g, CH
3COONa 3g, 10% soybean sprout (the long 4-5cm of bud boiled about 30 minutes) taking juice 1000ml, pH nature
(2) sterilization inoculation: boil sterilization in 5-10 minute, be cooled to and insert primary seed solution 5% about 35 ℃
(3) cultivate: copy the primary seed solution cultural method to cultivate 5-7 days
(4) index: similar to primary seed solution, but the bacterium viable count of cultivating descends 〉=1 * 10 to some extent
8Individual/ml, assorted bacterium rate 〉=5%
If three grades of seed liquor of preparation can be imitated the secondary seed solution preparation, only inoculum size can increase to 10-15%
The preparation of 3 compound seed liquid
3.1 culture medium prescription
(1) soybean sprout 100g (the long 4-5cm of bud boiled about 30 minutes) gets its juice 1000ml, in add oral glucose 75g, molasses (Bris is more than 80) 40-50g
(2) soybean sprout 100g (the long 4-5cm of bud boiled 30 minutes) gets its juice 1000ml, in add glucose 40g, CaCO
320g, fresh milk 2ml
3.2 sterilization, inoculation and cultivation
Boil sterilization, be cooled to 30 ℃, insert lactobacillus bulgaricus secondary or three grades of seed liquor 10% respectively, cereuisiae fermentum secondary or three grades of seed liquor 5%, under 28-33 ℃ of condition aerated culture 2-3 days
3.3 index: the bacterium of cultivating (or effective bacterium) viable count 〉=1 * 10
9Individual/ml, pH4-4.5, the sour-sweet wine flavour of tool, assorted bacterium rate≤5%
The production of 4AM microbial inoculum
4.1 nutrient solution prescription (%)
Soybean sprout 5-10 (the long 4-5 of bud, soybean sprout/water is 1/3, boils 40-50 minute) gets filtered juice 15-30, molasses (Bris is more than 80) 5, glucose 1.5-2, urea 0.25, CaCO
30.125-0.5, rare earth 0.08 (800ppm), N.F,USP MANNITOL 0.05 (500ppm), fresh milk 0.05 (500ppm), MgSO
47H
2O0.02 (200ppm), NaCl 0.02 (200ppm), KH
2PO
40.02 (200ppm), K
2HPO
40.02 (200ppm), FeSO
40.004 (40ppm), KNO
30.004 (40ppm), boric acid 0.004 (40ppm), ammonium molybdate 0.001 (10ppm)
4.2 sterilization, inoculation and cultivation
Quantitative water is boiled, add all quantitative composition, pH6-7.5, the plastics mash-back of packing into while hot, trickle are cooled to 35 ℃, inserted compound seed liquid 15-20% the same day respectively, EM stoste 5%, aerobic fermentation under the 30-35 ℃ of room temperature, the temperature of the fermented liquid of this moment is about 33 ℃, stirred ventilation 1 time, 2-3 minute/time bucket in per 4 hours.Aerobic fermentation 3-4 days, inserted rhodospirillum rubrum two or three grades of seed liquor 10% this moment, tightens lid immediately, sealing, carry out anaerobically fermenting, left standstill 8-12 days under 30-35 ℃, during constantly loosening fermentation bung emit fermentation flatulence, aerogenesis not to end, so far, product A M microbial inoculum is made
4.3 index
Living bacteria count 〉=1 * 10
8/ ml, pH value 2.9-4.1, assorted bacterium rate≤10%, intestinal bacteria MPN/1000ml≤30, fermented liquid is that the AM microbial inoculum is suspension, dark brown, tool alcohol tart flavour, organic acid total amount 1.56%, total amino acid content 380.2mg/ml also has materials such as VITAMIN and physiologically active.
The use of 5AM microbial inoculum
The using priciple of AM microbial inoculum product: require several method to be used, to be manured into soil, sprinkling is auxilliary, advocates and early executes, and requires crop to use the time of infertility, will obtain beyond thought effect; Can not mix with sterilant; When using city tap-water, should place aeration one day; This product seasoning is more than 5 ℃, keeps in Dark Place in normal temperature, drying, cool place, ventilation; This product fluid surface adularescent floss or the bottle end, have throw out all to belong to normally; In the bottle gas is arranged, note that when opening bottle with after please tighten the bottle cap preservation.
Concrete using method is summarized as follows:
5.1 filling root: dilution or water with water.During the seedling field planting, with 1: 500 times dilution liquid irrigating root, about every strain consumption 400-500 milliliter.
5.2 dip in root: this product is diluted by a certain percentage, add solid powders such as fertilizer, dip on root and cultivate.
5.3 fertilizer fermentation: is 1 by this product with organic fermented product: the mixed fermentation of 500-1000, make fermentation maturity, use as fertilizer or biological organic fertilizer, every 100-250kg/ mu, make base manure or topdress all can, but want soil to bury after using.
5.4 spray: 500-1000 doubly is sprayed directly on to the crop blade face with the dilution of this product.
5.5 seed soaking: with 1: 100-200 AM diluent doubly, seed soaking to swell (8-12 hour), are pulled out and are dried the back sowing.
5.6 seed dressing: the ratio of seed kind, this product, brown sugar, water is 1: 10-20: 10-20: an amount of, it is mixed thoroughly sowing (generally being used for the big crop of application rate) in vexed 2 hours.
5.7 bubble root: will be not during transplanting with the seedling root of soil 1: soak field planting in 15 minutes (flowers, nursery stock) in the 250-500 AM molasses diluent doubly.
The making of product activation solution: 10 kilograms in the water of 1 kilogram in this product stoste 1000ml+ brown sugar+cleaning, in the airtight container of packing into, under 25-35 ℃ condition anaerobically fermenting 3-7 days, to produce sour-sweet wine flavour, aerogenesis does not get final product., can not put for a long time with usefulness with system.
Above-mentioned AM microbial inoculum product provided by the invention, that selects to have determined already has the beneficial microorganism of promoter action to crop growthing development, with its mixed culture in same preparation, the a large amount of breedings in soil of lactobacillus bulgaricus, saccharomyces cerevisiae and rhodospirillum rubrum can make that beneficial microbe colony rolls up in the soil, thereby the growth and breeding that suppresses harmful bacterium such as spoilage organism alleviates or eliminates crop pest; The product of meta-bolites that probiotics produced and thalline itself can make soil improve crumb structure, increases organic nutrient, strengthens the soil comprehensive fertility, eliminates corrupt smell, for plant or crop provide direct nutrient, thereby lays a good foundation for high and stable yields; But effectively bacterial classification mutualism can form dominant microflora in the rhizospheric microflora of soil fauna, life-time service can cause the benign cycle of agriculture production environment; Effectively bacterial classification can decompose the agricultural chemicals residual in the soil and the toxic substance of accumulation, makes soil become reduced state.Thereby, this AM microbial inoculum product has the agriculture production environment of improvement, improve the effect of agricultural product quality and output, can be widely used in forestry (comprising fruit tree etc.), grass cultivation, flower industry, medium-height grass medicine company, cash crop (tealeaves, oil plant, cotton, fiber crops, cigarette etc.), so that in the farm crop industry multi-form agriculture such as (paddy and wheat, beans, potato, corn, sugarcane, vegetables etc.).
The preparation method of above-mentioned AM microbial inoculum provided by the invention according to its physiological property, correspondingly takes three kinds of selected effective mycorhiza scientifically to cultivate with the method that mixing, aerobic combine with anaerobism separately.Wherein: lactobacillus bulgaricus and cereuisiae fermentum are facultative anaerobe, have taked first aerobic to cultivate, so that its cell is bred fast and in large quantities; Carry out anaerobism then and cultivate, so that make institute's culturing cell further form effective meta-bolites.Rhodospirillum rubrum belongs to absolute anerobe, and light requirement need be cultivated under anaerobism and illumination condition all the time simultaneously, so that make it in propagated cell colony, produces effective meta-bolites.In same mixed-culture medium, rhodospirillum rubrum and cereuisiae fermentum growth must be obstructed, in order to guarantee in the product fermented liquid, to have enough effective meta-bolitess of these two kinds of bacterium, taked the advanced fermentation technique of the in good time by a certain percentage blended of selected effective bacterium bacterium liquid.Cereuisiae fermentum is acidproof, can in the acid nutrient solution of lactobacillus bulgaricus, grow, the fermented liquid of these two kinds of bacterium is at first mixed by a certain percentage, aerobic is cultivated, and treats that it entered anaerobically fermenting before the stage, added the rhodospirillum rubrum nutrient solution more by a certain percentage, carry out anaerobically fermenting, make that thalline is bred to greatest extent, meta-bolites is accumulated to greatest extent, and make its stable coexisting as in the same culture environment, so that the useful effect of common performance in agricultural environment.
For this reason, the present invention compared with prior art has following positively effect:
1. the contained effective strain effect of this microbial inoculum product is affirmed, effectively kind and viable count thereof are clear and definite, compared with similar products, has determined that clearly lactobacillus bulgaricus and cereuisiae fermentum for detecting index, have strengthened the operability of quality supervision and test.Living bacteria count 〉=1 * 10
8Individual/ml.Simultaneously, the contained effective detection bacterial classification of this product is provided by the Ministry of Agriculture microbial strains preservation administrative center, avoids can not reappearing phenomenon in the microbe to screen process, has strengthened the operability of bacterial classification utilization and detection.
2. effective culture of strains base or nutrient solution have been determined in screening, and cultural method.
3. take the training measures of first aerobic breeding thalline, back anaerobism stabilization formulations and chemical ingredients, explore a whole set of and produced the maturation process of this product, compared with similar products, aerobic, anaerobism, different strain synchronized culture have been overcome and the unbalanced phenomena that causes.1 to 2 year quality guaranteed period.
4. using method is versatile and flexible, and the aspects such as obnoxious flavour of burning the seedling phenomenon that cause that produce in improving the soil, promote crop growth, eliminating because of fertilizer fermenting raw materials processes such as living excrement have good effect.To improving crop quality, guaranteeing the tangible effect of crop high and stable yields tool.
5. the chemical ingredients in this product is comparatively clear and definite, and this also is one of advantage that is different from like product.
6. this product has been carried out the toxicity check, belonged to nontoxic level, this is also rare in like product.
7. this product is gathered materials on the spot, and material is easy to get, preparation method's strong operability, thereby cost is lower compared with similar products.
8. this product provides clear and definite test rating with the index of pathogenic bacterium as the assorted bacterium rate of check from product health and two aspects of product quality indicator.
Above-mentioned AM microbial inoculum may be summarized as follows at the effect of plantation aspect agriculture:
1. improve quality of agricultural product, improve the agricultural-food mouthfeel, recover genuine:, can make its effective constituent formic acid content improve 0.6% as on hops, using; On Wine Grape, use, can make the grape soluble solid content improve 2.6%.After using this microbial inoculum, wheat protein content improves 9%, and the peanut oil yield improves more than 8%, and sugarcane and high-quality muskmelon sugar degree improve 0.85-1.75 percentage point, and the cotton pulling force improves 5.8%, and Chinese cabbage Vc improves 24%, and fruit 1-2 level fruit rate improves 15-22%.
2. can improve the cash crop growth conditions: as on Chinese prickly ash, using, can make its plant with luxuriant foliage and spreading branches in leafy profusion, blade thickness, the leaf look dark green.Can make flowers prolong flowering time more than 1 times.Use on the flowers butterfly orchid, after 3 months, the flower that every strain is tied is increased to 20 by original 12.After red palm uses, from the Spring Festival to the end of the year, to bloom continuously always, leafiness, blade not only color are dark green, and leaf area reaches 30.20cm
2, untapped only 15.12cm
2
3. to increasing the loosing soil degree, the structure of improving the soil has positive effect: according to the experimental tests of Zhenyuan, Qingyang, make base manure in the Long Dong arid area with this microbial inoculum, pour with water, make the soil weight reduce 0.03-0.04g/cm
3, porosity increases 1.24-1.57%, and field capacity improves 1.2%.According to the Lanzhou experimental tests, on lanzhou lily, make seed manure, topdress and base fertilizer with this bacteria fermentation fertilizer, can make the loosing soil degree improve 45.24-86.5%, and the soil texture is poor more, effect is obvious more.
4. the high and stable yields effect is remarkable: as using on summer squash, can increase production 29%; On Radix Codonopsis, use, can increase production 18.2-33%; On cucumber, use, can increase production 18.9%.Use on food crop such as wheat, corn, amplification is respectively 8.5-11.5%, 11.5-23.5%; Can make bamboo shoot volume increase 10.9-21.2%, tomato volume increase 15.2-24.3% reaches as high as 33.3%, cucumber production promoting 18.9-27.3%, and summer squash volume increase 15.98-28.05%, the capsicum volume increase can reach 22.5%, and the winter rape volume increase reaches 19.1%; Use can improve the hay amount more than 15% on the grassland.According to various places test, demonstration, volume increase is in various degree arranged all on the various crops; Use on the cash crop flue-cured tobacco, leaf is long, and leaf is wide, Dan Yechong, and effectively the number of sheets all increases to some extent, can increase production 10.4-14.6%, every mu of pure income 47.6-69.7 unit that increases.
5. can impel crop products listing ahead of time: which kind of vegetables no matter, all than not using this microbial inoculum to go on the market in advance 7-9 days.Can make the flue-cured tobacco reduction in the life period 5-7 days.
But 6. pest control: the peaceful test point in Lanzhou, spray apple tree with the insect protected liquid of this fungicide preparation, be 90% to the efficient of preventing and treating of leaf roller, to the harm reduction by 5.3% of codling moth.
7. the AM microbial inoculum is being eliminated the foreign odor flavor, is suppressing assorted bacterium, is being prevented that aspect such as butt rot has certain effect.In the plastic greenhouse in Jingyuan County Ping Bao township, for a long time owing to generally use the organism such as sheep excrement, chicken manure, burning a heatable brick bed soil, the dregs of fat of fermentation maturity not, it is very serious to burn the seedling phenomenon.In this case, with the pouring of AM microbial inoculum diluent, the foreign odor flavor can disappear in second day.Crop plants such as injured cucumber, western blue melon, eggplant and capsicum take a turn for the better at once, burn the seedling phenomenon and disappear.This phenomenon explanation, burning seedling is NH
3And H
2Due to the irritant gass such as S, this microbial inoculum has deodorizing effect.Use in places such as lavatory, dormitories, receive the effect of tangible elimination peculiar smell equally.Generally there being bank up place or livestock and poultry colony house such as the ight soil of peculiar smell, domestic refuse to use, the positive effect of eliminating the foreign odor flavor is arranged all.
8. have a bacteria resistance function.In the test point, Zhenyuan County, on watermelon, test, root disease reduces 6% under the situation of low temperature, freeze injury.Result in the test point, Lintao shows, can prevent and treat cuiller and rot.In the test point, Lanzhou, with this microbial inoculum lily cuiller and seed ball are carried out immersion treatment after, to the control rate of its canker contrast improved 3-4 doubly.Above-mentioned phenomenon explanation, this microbial inoculum can also suppress spoilage organism, prevents and prevent the septic effect of crop root except general effect.
Specific implementation
Embodiment 1
1 single seed liquor preparation
1.1 the preparation of lactobacillus bulgaricus seed liquor
1.1.1 the preparation of primary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: peptone 2g, extractum carnis 3g, yeast extract paste 3.5g, glucose 40g, K
2HPO
40.5g, KH
2PO
40.5g, MgSO
47H
2O 0.5g, CaCO
32g, vitamin complex 0.01g (1) adds water to 1000ml, pH6.8.Be sub-packed in 5 500ml triangular flasks the 200ml/ bottle.
(2) sterilization and inoculation: conventional autoclaving, aseptic technique inoculation when being cooled to 35 ℃, (the female kind in 18 * 180mm) inclined-planes connects 5 triangular flasks to every test tube.
(3) cultivate: under the 35-38 ℃ of condition, 180 rev/mins of shaking culture 3 days.
(4) index: the bacterium viable count 1.59 * 10 of cultivating
8Individual/ml, pH4.5, tool yogurt milk fragrance.
1.1.2 the preparation of secondary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: glucose 50g, vitamin complex 50mg (50ppm), 10% soya bean (soybean) bud (boiling about 30 minutes) is got its juice and is added to 1000ml, the pH nature.Be sub-packed in 5 500ml triangular flasks the 200ml/ bottle.
(2) sterilization inoculation: boil sterilization in 5 minutes, when being cooled to 35 ℃, every triangular flask inserts primary seed solution 10ml (5%).
(3) cultivate: 35-40 ℃ of aerated culture 3 days, seal anaerobism then and cultivated 2 days, during constantly pine lid venting.
(4) index: the bacterium viable count 1.1 * 10 of cultivating
9Individual/ml, other index is with the one-level seed liquor.
1.2 the preparation of cereuisiae fermentum seed liquor
1.2.1 the preparation of primary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: new sweet wort (Bris 12) 1000ml, pH6.4.Be sub-packed in 5 500ml triangular flasks the 200ml/ bottle.
(2) disinfection inoculation: conventional autoclaving, aseptic technique when being cooled to 30 ℃, (the female kind in 18 * 180mm) inclined-planes connects 5 triangular flasks to every test tube.
(3) cultivate 28-32 ℃, 180 rev/mins of vibrations were cultivated 3 days.
(4) index: the bacterium viable count 1.83 * 10 of cultivating
7Individual/ml, pH5.8, tool wine flavour.
1.2.2 the preparation of secondary seed solution (is radix with 1000ml)
Copy above-mentioned first order seed liquid and preparation method thereof to amplify, difference is to boil sterilization, and every bottle of inoculum size increases to about 20ml (10%) aerated culture 2 days.
1.3 the preparation of rhodospirillum rubrum seed liquor
1.3.1 the preparation of primary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: peptone 2g, NH
4Cl 1g, K
2HPO
40.2g, CH
3COONa 3g, MgSO
47H
2O 0.2g, NaCl 1g, somatomedin liquid
(a)1ml (filtration sterilization, nutrient solution sterilization back adds), liquid microelement
(b)1ml adds water to 1000ml, and pH nature, average mark are packed in 5 300ml glass reagent bottles, the 200ml/ bottle.
(a) somatomedin liquid: vitamins B
110mg, Buddhist nun restrain butyric acid 1mg, para-amino benzoic acid 1mg, and vitamin H (IAA) 0.01mg, water is settled to 1000ml.
(b) liquid microelement: FeCl
350mg, CuSO
40.5mg, H
3PO
410ml, NaCl 20.5mg, ZnSO
410mg, CO (NO
3)
25mg, water is settled to 1000ml.
(2) disinfection inoculation: conventional autoclaving, be cooled to about 35 ℃ after, (the female kind in 18 * 180mm) inclined-planes connects 2.5 reagent bottles to per 1 test tube.
(3) cultivate: airtight, leave standstill, anaerobism is cultivated, and the 60W incandescent light shines on four sides.Incandescent light was cultivated 10 days for 36-38 ℃ apart from glass culturing bottle 10cm.
(4) index: nutrient solution changes scarlet into by khaki color, the bacterium viable count 1.36 * 10 of cultivating
9Individual/ml, pH6.5 has strong rotten-egg odour.
1.3.2 the preparation of secondary seed solution (is radix with 2000ml)
(1) nutrient solution prescription: NaCl 2g, NH
4Cl 2g, K
2HPO
40.4g, MgSO
47H
2O 0.4g, peptone 3.0g, CH
3COONa 6g, 10% soybean sprout (the long 4-5cm of bud boiled about 30 minutes) taking juice 2000ml, pH nature.Average mark is loaded in 4 500ml reagent bottles.
(2) sterilization inoculation: boil sterilization in 5-10 minute, when being cooled to 35 ℃, insert primary seed solution 25ml (5%) in each reagent bottle.
(3) cultivate: copy the primary seed solution cultural method to cultivate 7 days.
(4) index: similar to primary seed solution, living bacteria count 1.3 * 10
8Individual/ml.
2 compound seed liquid are equipped with (is radix with 2000ml)
2.1 culture medium prescription: soybean sprout 200g (the long 4-5cm of bud boiled about 30 minutes) gets its juice 2000ml, in add oral glucose 150g, molasses (Bris is more than 80) 90g.Be sub-packed in 8 500ml triangular flasks the 250ml/ bottle.
2.2 sterilization, inoculation and cultivation
Boil sterilization, be cooled to 30 ℃, insert lactobacillus bulgaricus secondary seed solution 25ml (10%) respectively, cereuisiae fermentum secondary seed solution 12.5ml (5%).Aerated culture is 3 days under 28-33 ℃ of condition.
2.3 index
Effective bacterium viable count 〉=1.83 * 10
9Individual/ml, pH4, the sour-sweet wine flavour of tool.
The production of 3AM microbial inoculum
3.1 nutrient solution prescription (is radix with 20L)
Soybean sprout juice 1000g (the long 4-5 of bud, soybean sprout/water is 1kg/3L, boils 40 minutes) gets filtered juice 3000ml, molasses (Bris is more than 80) 1000g, glucose 400g, urea 50g, CaCO
3100g, rare earth 16g, N.F,USP MANNITOL 10g, fresh milk 10g, MgSO
47H
2O 8g, NaCl 8g, KH
2PO
48g, K
2HPO
48g, FeSO
40.8g, KNO
30.8g, boric acid 0.8g, ammonium molybdate 0.2g.Water with the flushing soybean sprout adds to 20L.
3.2 sterilization, inoculation and cultivation
20L water is boiled, add all prescription compositions, pH7, average mark is loaded in 8 300ml plastic tanks while hot, the 250ml/ bucket.Trickle is cooled to 35 ℃, in each mash-back, inserted the above-mentioned compound seed liquid 37.5ml (15%) that has prepared the same day respectively, EM stoste 12.5ml (5%), room temperature is controlled at 30-35 ℃, bung covers with 6 layers of gauze of wringing out liquid behind 0.2% bromogeramine medicining liquid dipping, approximately intermittently rocks 1 time in per 4 hours, carries out aerobic fermentation, at this moment, the temperature of fermented liquid is about 33 ℃.Aerobic fermentation 3 days inserts the rhodospirillum rubrum secondary seed solution, and every bottle of 25ml (10%) tightens the fermentation bung at once, and anaerobically fermenting is carried out in sealing, left standstill 8 days under 30-35 ℃, during constantly loosening fermentation bung so that emit bulging gas, aerogenesis not to end.So far, product A M microbial inoculum can be accused and be made.
3.3 index
Living bacteria count 1.6 * 10
9/ ml, PH3.5, assorted bacterium rate 8%, microbial inoculum is suspension, dark brown, tool alcohol tart flavour.
4 using method and effect
Make the cultivation object with annual Radix Codonopsis seedling.Experimental plot area 45m
2(0.0675 mu), contrast 15m
2(0.0225 mu), 9 strains/m
2, the experimental plot applies through the farm manure 30.36kg of AM microbial inoculum composting (in 450kg/ mu) and makes base fertilizer, simultaneously respectively at seedling stage and flowering period respectively spraying the blade face once with 500 times of diluents of AM microbial inoculum; Handle with the farm manure (10.12kg) and the clear water of isodose in the contrast field.
Excavate the Radix Codonopsis root, the result shows after autumn: experimental plot (45m
2) obtain bright root 42.46kg (amounting to per mu yield 632kg), contrast field (15m
2) be 10.68kg (amounting to per mu yield 475.1kg), average every 15m
2The experimental plot than obtaining 3.53kg (amount to every mu and obtain 156.9kg more) with the homalographic check plot, volume increase 33.1% more.Simultaneously, the Radix Codonopsis leaf look in the experimental plot, plant height, stem are thick etc., and the production economic characters all obviously are better than contrast, and have positive correlation with the output of underground part (root).As seen, AM microbial inoculum tool production-increasing function (this test point is located at the mountain area, plateau of Wudu, Gansu height above sea level 2100m).
Embodiment 2
The single seed liquor preparation of l
1.1 the preparation of lactobacillus bulgaricus seed liquor
1.1.1 the preparation of primary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: peptone 10g, dibasic ammonium citrate 2g, yeast extract paste or yeast powder 5g, extractum carnis 10g, MgSO
47H
2O 0.2g, MnSO
40.05g, glucose 20g, sucrose 5g, lactose 5g, CH
2COONa5g, K
2HPO
42g, tween 80 1ml, water adds to 1000ml, pH6.4.Be sub-packed in 5 500ml triangular flasks the 200ml/ bottle.
(2) sterilization and inoculation: conventional autoclaving, sterilize down for 121 ℃ got final product in 15 minutes, when being cooled to 35 ℃, the aseptic technique inoculation, (the female kind in 18 * 180mm) inclined-planes connects 5 culture jars to every test tube.
(3) cultivate: under the 35-38 ℃ of condition, 180 rev/mins of shaking culture 2 days.
(4) index: the bacterium viable count 1.2 * 10 of cultivating
8Individual/ml, pH4, tool yogurt milk fragrance.
1.1.2 the preparation of secondary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: glucose 50g, vitamin complex 50mg (50ppm), 10% soya bean (soybean) bud (boiling about 30 minutes) is got its juice and is added to 1000ml, the pH nature.Boil sterilization in 8 minutes, be sub-packed in 5 500ml triangular flasks the 200ml/ bottle while hot.
(2) inoculation: when being cooled to 35 ℃, every bottle graft is gone into primary seed solution 10ml (5%).
(3) cultivate: 35-40 ℃, aerated culture 3 days seals anaerobism then and cultivated 2 days, during constantly pine lid venting.
(4) index: bacterium viable count 〉=1.32 * 10 of cultivating
9Individual/ml, other index is with the one-level seed liquor.
1.2 the preparation of cereuisiae fermentum seed liquor
1.2.1 the preparation of primary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: soybean sprout 100g, (boiling about 30 minutes) gets its juice constant volume 1000ml, wherein adds glucose 50g, pH6.5.Average mark is loaded in 5 500ml triangular flasks, the 200ml/ bottle.
(2) disinfection inoculation: conventional autoclaving, aseptic technique when being cooled to 30 ℃, every test tube (5 culture jars of the female kind in 18 * 180mm) inclined-planes.
(3) cultivate: 28-32 ℃, 190 rev/mins of vibrations were cultivated 2 days.
(4) index: the bacterium viable count 1.6 * 10 of cultivating
7Individual/ml, pH5.8, tool wine flavour.
1.2.2 the preparation of secondary seed solution (is radix with 1000ml)
Copy above-mentioned first order seed liquid and preparation method thereof to amplify, difference is to boil sterilization, meets primary seed solution 20ml (10%) in each triangular flask, rocks aerated culture.
1.3 the preparation of rhodospirillum rubrum seed liquor
1.3.1 the preparation of primary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: peptone 2g, NH
4Cl 1g, K
2HPO
40.2g, CH
3COONa 3g, MgSO
47H
2O 0.2g, NaCl 1g, somatomedin liquid
(a)1ml (filtration sterilization, nutrient solution sterilization back adds), liquid microelement
(b)1ml adds water to 1000ml, and the pH nature is sub-packed in 4 300ml glass reagent bottles the 250ml/ bottle.
(a) somatomedin liquid: vitamins B
110mg, Buddhist nun restrain butyric acid 1mg, para-amino benzoic acid 1mg, and vitamin H (IAA) 0.01mg, water is settled to 1000ml.
(b) liquid microelement: FeCl
350mg, CuSO
40.5mg, H
3PO
410ml, NaCl 20.5mg, ZnSO
410mg, CO (NO
3)
25mg, water is settled to 1000ml.
(2) disinfection inoculation: conventional autoclaving, be cooled to about 35 ℃ after, (18 * 180mm) inclined-planes are female plants, and every connects 2 bottles to insert test tube.
(3) cultivate: airtight, leave standstill, anaerobism is cultivated, and the 60W incandescent light shines on four sides.Incandescent light was cultivated 13 days for 36-38 ℃ apart from glass culturing bottle 12cm.
(4) index: nutrient solution changes scarlet into by blush, the bacterium viable count 1.85 * 10 of cultivating
9Individual/ml, pH7.2 has strong rotten-egg odour.
1.3.2 the preparation of secondary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: NaCl 1g, NH
4Cl 1g, K
2HPO
40.2g, MgSO
47H
2O 0.2g, peptone 1.5g, CH
3COONa 3g, 10% soybean sprout (the long 4-5cm of bud boiled about 30 minutes) taking juice 1000ml, pH nature.Be sub-packed in 4 300ml glass reagent bottles the 250ml/ bottle.
(2) sterilization inoculation: boil sterilization in 5-10 minute, be cooled to about 35 ℃, every bottle graft is gone into primary seed solution 12.5ml (5%).
(3) cultivate: copy the primary seed solution cultural method to cultivate 6 days.
(4) index: similar to primary seed solution, the bacterium viable count 1.1 * 10 of cultivating
8Individual/ml.
2 compound seed liquid are equipped with (is radix with 1000ml)
2.1 culture medium prescription: soybean sprout 100g (the long 4-5cm of bud boiled about 30 minutes) gets its juice 1000ml, in add glucose 40g, CaCO
320g, fresh milk 2ml.Boil sterilization, in 5 the 300ml triangular flasks of packing into while hot, the 200ml/ bottle.
2.2 inoculation and cultivation
Be cooled to 30 ℃, every bottle is inserted lactobacillus bulgaricus secondary seed solution 20ml (10%) respectively, and cereuisiae fermentum secondary seed solution 10ml (5%) swayed aerated culture 3 days under 30 ℃ of conditions.
2.3 index: effective bacterium viable count 1.4 * 10
9Individual/ml, pH4.5, the sour-sweet wine flavour of tool.
The production of 3AM microbial inoculum
3.1 nutrient solution prescription (is radix with 20L)
Soybean sprout juice 1600g (the long 4-5 of bud, soybean sprout/water is 1600g/4800ml, boils 45 minutes) gets filtered juice 4800ml, molasses (Bris is more than 80) 1000g, glucose 360g, urea 50g, CaCO
350g, rare earth 16g, N.F,USP MANNITOL 10g, fresh milk 10g, MgSO
47H
2O 4g, NaCl 4g, KH
2PO
44g, K
2HPO
44g, FeSO
40.8g, KNO
30.8g, boric acid 0.8g, ammonium molybdate 0.2g.
3.2 sterilization, inoculation and cultivation
20L water is boiled, add all prescription compositions, pH6.5, on average pack into while hot in 10 250ml plastic tanks, trickle is cooled to 35 ℃, inserts above-mentioned lactobacillus bulgaricus that has prepared and saccharomyces cerevisiae compound seed liquid 17% same day respectively, EM stoste 5%, cultivate under 33 ℃ of room temperatures, unscrew bung, stirred 1 time in per 4 hours, 3 minutes/time buckets, make its aerobic fermentation, at this moment, the temperature of fermented liquid is controlled to be about 33 ℃.Behind the aerobic fermentation 4 days, insert the rhodospirillum rubrum secondary seed solution 10% that has prepared, the circular rubber packing that is of moderate size on the pad in the bung, tighten bung, anaerobically fermenting is carried out in sealing, left standstill under 33 ℃ 10 days, during constantly loosening fermentation bung emit fermentation flatulence, aerogenesis not to end.So far, product A M microbial inoculum is made.
3.3 index: living bacteria count 1.5 * 10
8/ ml, pH3.6 is suspension, dark brown, tool alcohol tart flavour.
4 using method and effect
Make test materials with 500 strains biennial " sodium Gai Te " hops.With AM microbial inoculum 1L, with brown sugar 1kg, it is an amount of to add water, admixes 1m
3In the cow dung, heap fermentation 20 days, to stink eliminate, quality soft till, make AM bacteria fermentation biological organic fertilizer.With 500 times of this biological organic fertilizer dilutions, respectively at May 20 calendar year 2001 and June 1 irritating root 1 time, 1000ml/ strain uses the cow dung of handling without the AM bacteria fermentation to do corresponding contrast simultaneously, contrasts 50 strains.The hops inflorescence of gathering mid-September was delivered to Gansu Province plant nutrition institute of land-reclaimable farming research institute laboratory and is adopted conductometric titration to chemically examine on October 10.The result shows, at conversion moisture content is on 8% the basis, handle sample and contain alpha-acid (formic acid) 13.40%, and contrast to contain 12.87%, handles comparison according to having increased by 0.53%.Alpha-acid is the main effective constituent of hops inflorescence, is a leading indicator judging hops quality quality.As seen, AM microbial inoculum tool improves or improves the effect (this test point is located at Wuwei Mongolian gazelle town, Gansu) of crop quality.
Embodiment 3
1 single seed liquor preparation
1.1 the preparation of lactobacillus bulgaricus seed liquor
1.1.1 the preparation of primary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: peptone 2g, extractum carnis 3g, yeast extract paste 3.5g, glucose 40g, K
2HPO
40.5g, KH
2PO
40.5g, MgSO
47H
2O 0.5g, CaCO
32g, vitamin complex 0.01g (1) adds water to 1000ml, pH6.8.Be sub-packed in 5 500ml triangular flasks the 200ml/ bottle.
(2) sterilization and inoculation: conventional autoclaving, aseptic technique inoculation when being cooled to 35 ℃, (the female kind in 18 * 180mm) inclined-planes connects 5 above-mentioned nutrient solution triangular flasks to every test tube.
(3) cultivate: under the 35-38 ℃ of condition, 180 rev/mins of vibrations were cultivated 3 days.
(4) index: the bacterium viable count 1.5 * 10 of cultivating
8Individual/ml, pH5, tool yogurt milk fragrance.
1.1.2 the preparation of secondary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: glucose 50g, vitamin complex 50mg, 10% soya bean (soybean) bud boiled about 30 minutes, got its juice and added to 1000ml, the pH nature.Be sub-packed in 5 500ml triangular flasks the 200ml/ bottle.
(2) sterilization inoculation: boil sterilization in 10 minutes, when being cooled to 35 ℃, each triangular flask inserts primary seed solution 10ml (5%).
(3) cultivate: 35-40 ℃ of aerated culture 3 days, seal anaerobism then and cultivated 3 days, during constantly pine lid venting.
(4) index: the bacterium viable count 1.6 * 10 of cultivating
9Individual/ml, other index is with the one-level seed liquor.
1.2 the preparation of cereuisiae fermentum seed liquor
1.2.1 the preparation of primary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: measure new sweet wort (Bris 12) 1000ml, pH6.4.Be sub-packed in 4 500ml triangular flasks the 250ml/ bottle.
(2) disinfection inoculation: conventional autoclaving, aseptic technique when being cooled to 30 ℃, (the female kind in 18 * 180mm) inclined-planes connects 4 triangular flasks to every test tube.
(3) cultivate 28-32 ℃, 200 rev/mins of vibrations were cultivated 3 days.
(4) index: the bacterium viable count 1.9 * 10 of cultivating
7Individual/ml, pH5.8, tool wine flavour.
1.2.2 the preparation of secondary seed solution (is radix with 1000ml)
Copy above-mentioned first order seed liquid and preparation method thereof to amplify, the 1000ml nutrient solution is sub-packed in 4 500ml triangular flasks the 250ml/ bottle.Difference is to boil sterilization, and every bottle graft primary seed solution 25ml (about 10%) rocks aerated culture.
1.3 the preparation of rhodospirillum rubrum seed liquor
1.3.1 the preparation of primary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: peptone 2g, NH
4Cl 1g, K
2HPO
40.2g, CH
3COONa 3g, MgSO
47H
2O 0.2g, NaCl 1g, somatomedin liquid
(a)1ml (filtration sterilization, nutrient solution sterilization back adds), liquid microelement
(b)1ml adds water to 1000ml, and the pH nature is in the Glass Containers of packing into.
(a) somatomedin liquid: vitamins B
110mg, Buddhist nun restrain butyric acid 1mg, para-amino benzoic acid 1mg, and vitamin H (IAA) 0.01mg, water is settled to 1000ml.
(b) liquid microelement: FeCl
350mg, CuSO
40.5mg, H
3PO
410ml, NaCl 20.5mg, ZnSO
410mg, CO (NO
3)
25mg, water is settled to 1000ml.Be sub-packed in 5 200ml transparent glass reagent bottles the 200ml/ bottle.
(2) disinfection inoculation: conventional autoclaving, be cooled to about 35 ℃ after, (the female kind in 18 * 180mm) inclined-planes connects above-mentioned 2.5 reagent bottles for every test tube.
(3) cultivate: airtight, leave standstill, anaerobism is cultivated, and the 40W incandescent light shines on four sides.Incandescent light was cultivated 15 days for 36-38 ℃ apart from glass culturing bottle 10cm.
(4) index: nutrient solution changes scarlet into by khaki color, the bacterium viable count 1.76 * 10 of cultivating
9Individual/ml, pH6.7 has strong rotten-egg odour.
1.3.2 the preparation of secondary seed solution (is radix with 1000ml)
(1) nutrient solution prescription: NaCl 1g, NH
4Cl 1g, K
2HPO
40.2g, MgSO
47H
2O 0.2g, peptone 1.5g, CH
3COONa 3g, 10% soybean sprout (the long 4-5cm of bud boiled about 30 minutes) taking juice 1000ml, pH nature.Boil sterilization in 10 minutes, be sub-packed in 5 200ml transparent glass reagent bottles the 200ml/ bottle while hot.
(2) inoculation: be cooled to about 35 ℃, every bottle graft is gone into primary seed solution 10ml (5%).
(3) cultivate: copy the primary seed solution cultural method to cultivate 6 days.
(4) index: similar to primary seed solution, the bacterium viable count 1.1 * 10 of cultivating
8Individual/ml.
2 compound seed liquid are equipped with (is radix with 1000ml)
2.1 culture medium prescription: soybean sprout 100g (the long 4-5cm of bud boiled about 30 minutes) gets its juice 1000ml, in add oral glucose 75g, molasses (Bris is more than 80) 50g.Boil sterilization in 10 minutes, be sub-packed in 5 200ml transparent glass reagent bottles the 200ml/ bottle while hot.
2.2 inoculation and cultivation: be cooled to 30 ℃, insert lactobacillus bulgaricus secondary seed solution 20ml (10%) respectively, cereuisiae fermentum secondary seed solution 10ml (5%).Under 28-33 ℃ of condition, rock, aerated culture 3 days.
2.3 index
The bacterium viable count 1.6 * 10 of cultivating
9Individual/ml, pH4.5, the sour-sweet wine flavour of tool, assorted bacterium rate 4.5%.
The production of 3AM microbial inoculum
3.1 nutrient solution prescription (is radix with 20L)
Soybean sprout juice 2000g (the long 4-5 of bud, soybean sprout/water is 2000g/6000ml, boils 50 minutes) gets filtered juice 6000ml, molasses (Bris is more than 80) 1000g, glucose 300g, urea 50g, CaCO
325g, rare earth 16g, N.F,USP MANNITOL 10g, fresh milk 10ml, MgSO
47H
2O 4g, NaCl 4g, KH
2PO
44g, K
2HPO
44g, FeSO
40.8g, KNO
30.8g, boric acid 0.8g, ammonium molybdate 0.2g.Water with the flushing soybean sprout adds to 20L.
3.2 sterilization, inoculation and cultivation
20L water is boiled, add all prescription compositions, pH6.5, average mark is loaded in 8 300ml plastic tanks while hot, the 250ml/ bucket.Trickle is cooled to 35 ℃, in each mash-back, inserted the above-mentioned compound seed liquid 37.5ml (15%) that has prepared the same day respectively, EM stoste 12.5ml (5%), room temperature is controlled at 30-35 ℃, bung covers with 6 layers of gauze of wringing out liquid behind 0.2% bromogeramine medicining liquid dipping, approximately intermittently rocks 1 time in per 4 hours, carries out aerobic fermentation, at this moment, the temperature of fermented liquid is about 33 ℃.Aerobic fermentation 3-4 days, inserted the rhodospirillum rubrum secondary seed solution this moment, and every bottle of 25ml (10%) tightens the fermentation bung at once, anaerobically fermenting is carried out in sealing, leaves standstill 12 days under 30-35 ℃, constantly loosening during this time fermentation bung, so that emit bulging gas, aerogenesis not to end.So far, product A M microbial inoculum can be accused and be made.
3.3 index
Living bacteria count 1.2 * 10
8Individual/ml, pH3.5 is suspension, dark brown, tool alcohol tart flavour.
4 using method and effect
AM microbial inoculum seed soaking cultivating method and effect test (test site is selected in Hui County, area, Wudu, Gansu Province, April 16 calendar year 2001) have following result:
(1) treatment process: add 150 times of diluent soaking corns of equivalent brown sugar seed 12-14 hour with the AM microbial inoculum, liquid level exceeds corn seed 10cm, till the seed swelling.Pull out and dry in the shade, sowing on the same day.Experimental plot area 252m
2, broadcast 90 row altogether, line-spacing 85cm, every row 10 strains, spacing in the rows 33cm.
Contrast is handled with 150 times of diluents of brown sugar, contrast field area 126m
2
(2) result: species test on October 12 then, number of grain per ear and thousand seed weight such as following table (on average)
| Grain number/fringe (grain) | Thousand seed weight (g) | Survey and produce (kg/ mu) | Organoleptic feature (by seedling stage to trumpet period) | |
| Contrast | 517.3 | 519.0 | 878 | Seedling is yellowish green, and carefully short, leaf was crispaturaed in the dry period, and black sick and Pyrausta nubilalis (Hubern). disease and pest takes place |
| Handle | 632.5 | 599.0 | 1102 | Seedling is dark green, and robust plant is tall and big, drought resisting, the effect of disease and pests such as tangible smut resistance of tool and Pyrausta nubilalis (Hubern). |
| Rate of increase (%) | 22.3％ | 15.4 | 25.5 |
This shows the tangible effect of increasing production of AM microbial inoculum seed soaking tool.
Claims (3)
1, a kind of agriculture production environment that improves, improve the microbial inoculum of agricultural product quality and output, it is characterized in that effective bacterial classification that this microbial inoculum is selected for use is lactobacillus bulgaricus (Lactobacillus bulgaricus) and cereuisiae fermentum (Saccharomyces cerevisiare) and rhodospirillum rubrum (Rhodospirillum rabrum), the above two are provided by the Ministry of Agriculture microbial strains preservation administrative center, and (sample number into spectrum is respectively: IFFI 6047, ACCC 2038), the back is got a kind of the separation from soil according to routine techniques by the applicant, with three kinds of selected effective mycorhiza according to its physiological property, correspondingly take separately and mix, the method that aerobic combines with anaerobism is scientifically cultivated, obtain thus a kind of have imitate bacterial classification and its meta-bolites is the fermentation preparation of one, the contained effective total viable count of microbial inoculum 〉=1 * 10
8Individual/ml.
2, a kind of preparation method who improves agriculture production environment according to claim 1, improves the microbial inoculum of agricultural product quality and output is characterized in that it may further comprise the steps:
(1) the female kind in test tube slant prepares
When preparation lactobacillus bulgaricus test tube slant mother plants, adopt with glucose, CaCO
3And soybean sprout juice is main substratum; When preparation cereuisiae fermentum test tube slant mother plants, adopt the substratum based on glucose, peptone, yeast extract paste and maltose, the female kind in the test tube slant of the two all prepares by ordinary method
When preparation rhodospirillum rubrum test tube slant mother planted, its substratum adopted with NaCl, CH
3COONa, NH
4Cl, NaHCO
3, K
2HPO
4, MgSO
4.7H
2O and somatomedin liquid and liquid microelement are main prescription, take the method illumination cultivation with the whiteruss sealing
(2) single seed liquor preparation
(1) preparation of lactobacillus bulgaricus seed liquor
During the preparation primary seed solution, its nutrient solution is selected for use with glucose, yeast extract paste, extractum carnis, peptone, CaCO
3And inorganic salt are main prescription, or select for use with glucose, peptone, extractum carnis, yeast extract paste or yeast powder, sucrose, lactose, CH
2COONa, dibasic ammonium citrate, tween 80 and inorganic salt are main prescription, conventional aseptic technique inoculation, and shaking culture is treated viable count in the nutrient solution 〉=1 * 10
8Individual/ml, pH3.8-5.4 get final product during tool yogurt milk fragrance simultaneously
The preparation of secondary seed solution: nutrient solution adopts the prescription based on glucose, soya bean/big bean sprouts juice, boil sterilization, conventional aseptic technique, insert primary seed solution 5%, aerated culture at first, seal anaerobism then and cultivate, during constantly put the flatulence of fermenting, treat to get final product when nutrient solution is similar to primary seed solution
If need three grades of seed liquor, except that bacterial classification is that secondary seed solution, inoculum size increase to the 7-10%, other is with the preparation of secondary seed solution
(2) preparation of cereuisiae fermentum seed liquor
The preparation of primary seed solution: nutrient solution is selected the prescription based on new sweet wort Bris 12 or glucose and soybean sprout juice for use, conventional aseptic technique, and inoculation, shaking culture is treated viable count 〉=1 * 10 in the nutrient solution
7Individual/ml, pH4-5.8 get final product during the tool wine flavour simultaneously
Two---the preparation of three grades of seed liquor: copy above-mentioned first order seed liquid and preparation method thereof to amplify, difference is to boil sterilization, connects one-level or previous stage seed liquor about 10%, aerated culture
(3) preparation of rhodospirillum rubrum seed liquor
The preparation of primary seed solution: nutrient solution is selected for use with CH
3COONa, peptone, NH
4Cl, NaCl and inorganic salt, somatomedin liquid and liquid microelement are main prescription, in the Glass Containers of packing into, and conventional aseptic technique, inoculation, leave standstill, incandescent light shines on four sides, and closing anaerobic is cultivated, and treats that nutrient solution changes scarlet, bacterium viable count 〉=1 * 10 of cultivating into by original khaki color
9Individual/ml, pH is near neutral, and gets final product when having strong rotten-egg odour
The preparation of secondary seed solution: nutrient solution is selected for use with CH
3COONa, peptone, soybean sprout juice, NaCl, NH
4Cl and some other inorganic salt are main prescription, boil sterilization, and conventional aseptic technique inoculation inserts primary seed solution 5%, copies the primary seed solution cultural method to cultivate, and treats to get final product when nutrient solution is similar to primary seed solution
If need three grades of seed liquor of preparation, can imitate the secondary seed solution preparation, only inoculum size can increase to 10-15%
(3) compound seed liquid preparation
Substratum adopts based on oral glucose, the molasses of Bris more than 80 and the prescription of soybean sprout juice; Or adopt to add the prescription of glucose and fresh milk in the soybean sprout juice, boil sterilization, insert lactobacillus bulgaricus secondary or three grades of seed liquor 10%, cereuisiae fermentum secondary or three grades of seed liquor 5% respectively, aerated culture is treated effective bacterium viable count 〉=1 * 10 in the nutrient solution
9Individual/ml, pH4-4.5, and get final product during the sour-sweet wine flavour of tool
(4) preparation of AM microbial inoculum product or production
Nutrient solution is made up of following ingredients: soybean sprout filtered juice 15-30%, Bris 80 above molasses 5%, glucose 1.5-2%, urea 0.25%, and CaCO
3, rare earth, N.F,USP MANNITOL, fresh milk, several micro-inorganic salt have also been added in addition, boil sterilization, the plastics mash-back of packing into while hot, trickle is cooled to 35 ℃, inserts compound seed liquid 15-20%, EM stoste 5% same day respectively, cultivates under 30-35 ℃ of room temperature and aerobic conditions, after about 3-4 days, insert rhodospirillum rubrum two or three grades of seed liquor 10%, tighten the fermentation bung at once, sealing, carrying out anaerobism cultivates, left standstill under 30-35 ℃ 8-12 days, during constantly loosening fermentation bung so that emit fermentation flatulence, aerogenesis not to end, so far, product A M microbial inoculum is made.
3,, it is characterized in that they are below concrete steps according to the described bacterial preparation process of claim 2:
The female kind in 1 test tube slant prepares
1.1 female kind the in lactobacillus bulgaricus test tube slant prepares
(1) culture medium prescription: glucose 40g, CaCO
320g milk 2ml, agar 18-20g boils and gets 10% soybean sprout juice and be settled to 1000ml, is sub-packed in the test tube
(2) sterilization, inoculation and cultivate: conventional autoclaving, 30-33 ℃ of cultivation of inoculation 5-7 days smeared in pendulum inclined-plane aseptic technique, covers with the inclined-plane to lawn
1.2 female kind the in cereuisiae fermentum test tube slant prepares
(1) culture medium prescription: maltose 1.5g, yeast extract paste 3g; Peptone 5g, glucose 10g, agar 15-18g, water adds to 1000ml, is sub-packed in the test tube
(2) sterilization, inoculation and cultivate: similar to the female preparation method of kind of above-mentioned Bulgaria, but 28-32 ℃ of cultivation 2-3 days treated to be covered with on the inclined-plane white lawn and got final product
1.3 female kind the in rhodospirillum rubrum test tube slant prepares
(1) culture medium prescription: NaCl 0.5-2g, NH
4Cl 1g, NaHCO
31g (filtration sterilization), K
2HPO
40.2g, MgSO
47H
2O 0.2g, CH
3COONa 1-5g, somatomedin liquid
(a)1ml (filtration sterilization, nutrient solution sterilization back adds), liquid microelement
(b)1ml, agar 15-18g adds water to 1000ml, and the pH nature is sub-packed in the test tube
(a) somatomedin liquid: vitamins B
110mg, Buddhist nun restrain butyric acid 1mg, para-amino benzoic acid 1mg, and vitamin H (IAA) 0.01mg, water is settled to 1000ml
(b) liquid microelement: FeCl
350mg, CuSO
40.5mg, H
3PO
410ml, NaCl 20.5mg, ZnSO
410mg, CO (NO
3)
25mg, water is settled to 1000ml
(2) sterilization, inoculation: prepare similarly to female kind of above-mentioned lactobacillus bulgaricus, but with the whiteruss sealing, 30-35 ℃, illumination (5000-10000lx) was cultivated 14-50 days, treated to occur on the inclined-plane red lawn and got final product
2 single seed liquor preparations
2.1 the preparation of lactobacillus bulgaricus seed liquor
2.1.1 the preparation of primary seed solution
(1) nutrient solution prescription
1. peptone 2g, extractum carnis 3g, yeast extract paste 3.5g, glucose 40g, K
2HPO
40.5g, KH
2PO
40.5g, MgSO
47H
2O 0.5g, CaCO
32g, vitamin complex 0.01g (1) adds water to 1000ml, pH6.8
2. peptone 10g, dibasic ammonium citrate 2g, yeast extract paste or yeast powder 5g, extractum carnis 10g, MgSO
47H
2O 0.2g, MnSO
40.05g, glucose 20g, sucrose 5g, lactose 5g, CH
2COONa5g, K
2HPO
42g, tween 80 1ml, water adds to 1000ml, and pH6.4 sterilized 15 minutes down for 121 ℃
(2) sterilization and inoculation: conventional autoclaving, aseptic technique inoculation when being cooled to 35 ℃, every 1000ml nutrient solution connects test tube, and (18 * 180mm) inclined-planes are female plants 1
(3) cultivate: under the 35-38 ℃ of condition, 180 rev/mins shaking culture 2-3 days
(4) index: bacterium viable count 〉=1 * 10 of cultivating
8Individual/ml, pH3.8-5.4, tool yogurt milk fragrance
2.1.2 the preparation of secondary seed solution
(1) nutrient solution prescription: glucose 50g, vitamin complex 50mg (50ppm), 10% soya bean (soybean) bud boiled about 30 minutes, got its juice and added to 1000ml, the pH nature
(2) sterilization inoculation: boil sterilization in 5-10 minute, insert primary seed solution 5% when being cooled to 35 ℃
(3) cultivate: 35-40 ℃, aerated culture 2-3 days, seal anaerobism then and cultivated 2-3 days, during constantly pine lid venting
(4) index: bacterium viable count 〉=1 * 10 of cultivating
9Individual/ml, other index is with the one-level seed liquor
If need three grades of seed liquor, except that bacterial classification is that secondary seed solution, inoculum size increase to the 7-10%, other is with the preparation of secondary seed solution
2.2 the preparation of cereuisiae fermentum seed liquor
2.2.1 the preparation of primary seed solution
(1) nutrient solution prescription
1. new sweet wort (Bris 12), pH5-6.4
2. soybean sprout 100g (boiling about 30 minutes) gets its juice constant volume 1000ml, wherein adds glucose 50g, pH6-7
(2) disinfection inoculation: conventional autoclaving, aseptic technique when being cooled to 30 ℃, every 1000ml nutrient solution inserts test tube, and (18 * 180mm) inclined-planes are female plants 1
(3) cultivate 28-32 ℃, 180-200 rev/min shaking culture 1-3 days
(4) index: bacterium viable count 〉=1 * 10 of cultivating
7Individual/ml, pH4-5.8, tool wine flavour
2.2.2 two---the preparation of three grades of seed liquor
Copy above-mentioned first order seed liquid and preparation method thereof to amplify, difference is to boil sterilization, connects one-level or previous stage seed liquor about 10%, aerated culture
2.3 the preparation of rhodospirillum rubrum seed liquor
2.3.1 the preparation of primary seed solution
(1) nutrient solution prescription: peptone 2g, NH
4Cl 1g, K
2HPO
40.2g, CH
3COONa 3g, MgSO
47H
2O 0.2g, NaCl 1g, somatomedin liquid
(a)1ml (filtration sterilization, nutrient solution sterilization back adds), liquid microelement
(b)1ml adds water to 1000ml, and the pH nature is in the Glass Containers of packing into
(a) somatomedin liquid: vitamins B
110mg, Buddhist nun restrain butyric acid 1mg, para-amino benzoic acid 1mg, and vitamin H (IAA) 0.01mg, water is settled to 1000ml
(b) liquid microelement: FeCl
350mg, CuSO
40.5mg, H
3PO
410ml, NaCl 20.5mg, ZnSO
410mg, CO (NO
3)
25mg, water is settled to 1000ml
(2) disinfection inoculation: conventional autoclaving, be cooled to about 35 ℃ after, every 1000ml inserts test tube, and (18 * 180mm) inclined-planes are female plants 2
(3) cultivate: airtight, leave standstill, anaerobism is cultivated, and the 40-60W incandescent light shines on four sides.Incandescent light 36-38 ℃, was cultivated 10-15 days apart from glass culturing bottle 10-15cm
(4) index: nutrient solution changes scarlet into by khaki color, bacterium viable count 〉=1 * 10 of cultivating
9Individual/ml, pH has strong rotten-egg odour near neutral
2.3.2 the preparation of secondary seed solution
(1) nutrient solution prescription: NaCl 1g, NH
4Cl 1g, K
2HPO
40.2g, MgSO
47H
2O 0.2g, peptone 1.5g, CH
3COONa 3g, 10% soybean sprout (the long 4-5cm of bud boiled about 30 minutes) taking juice 1000ml, pH nature
(2) sterilization inoculation: boil sterilization in 5-10 minute, be cooled to and insert primary seed solution 5% about 35 ℃
(3) cultivate: copy the primary seed solution cultural method to cultivate 5-7 days
(4) index: similar to primary seed solution, but the bacterium viable count of cultivating descends 〉=1 * 10 to some extent
8Individual/ml, assorted bacterium rate 〉=5%
If three grades of seed liquor of preparation can be imitated the secondary seed solution preparation, only inoculum size can increase to 10-15%
The preparation of 3 compound seed liquid
3.1 culture medium prescription
(1) soybean sprout 100g (the long 4-5cm of bud boiled about 30 minutes) gets its juice 1000ml, in add oral glucose 75g, molasses (Bris is more than 80) 40-50g
(2) soybean sprout 100g (the long 4-5cm of bud boiled 30 minutes) gets its juice 1000ml, in add glucose 40g, CaCO
320g, fresh milk 2ml
3.2 sterilization, inoculation and cultivation
Boil sterilization, be cooled to 30 ℃, insert lactobacillus bulgaricus secondary or three grades of seed liquor 10% respectively, cereuisiae fermentum secondary or three grades of seed liquor 5%, under 28-33 ℃ of condition aerated culture 2-3 days
3.3 index: the bacterium of cultivating (or effective bacterium) viable count 〉=1 * 10
9Individual/ml, pH4-4.5, the sour-sweet wine flavour of tool, assorted bacterium rate≤5%
The production of 4AM microbial inoculum
4.1 nutrient solution prescription (%)
Soybean sprout 5-10 (the long 4-5 of bud, soybean sprout/water is 1/3, boils 40-50 minute) gets filtered juice 15-30, molasses (Bris is more than 80) 5, glucose 1.5-2, urea 0.25, CaCO
30.125-0.5, rare earth 0.08 (800ppm), N.F,USP MANNITOL 0.05 (500ppm), fresh milk 0.05 (500ppm), MgSO
47H
2O0.02 (200ppm), NaCl 0.02 (200ppm), KH
2PO
40.02 (200ppm), K
2HPO
40.02 (200ppm), FeSO
40.004 (40ppm), KNO
30.004 (40ppm), boric acid 0.004 (40ppm), ammonium molybdate 0.001 (10ppm)
4.2 sterilization, inoculation and cultivation
Quantitative water is boiled, add all quantitative composition, pH6-7.5, the plastics mash-back of packing into while hot, trickle are cooled to 35 ℃, inserted compound seed liquid 15-20% the same day respectively, EM stoste 5%, aerobic fermentation under the 30-35 ℃ of room temperature, the temperature of the fermented liquid of this moment is about 33 ℃, stirred ventilation 1 time, 2-3 minute/time bucket in per 4 hours.Aerobic fermentation 3-4 days, inserted rhodospirillum rubrum two or three grades of seed liquor 10% this moment, tightens lid immediately, sealing, carry out anaerobically fermenting, left standstill 8-12 days under 30-35 ℃, during constantly loosening fermentation bung emit fermentation flatulence, aerogenesis not to end, so far, product A M microbial inoculum is made
4.3 index
Living bacteria count 〉=1 * 10
8/ ml, pH value 2.9-4.1, assorted bacterium rate≤10%, intestinal bacteria MPN/1000ml≤30, fermented liquid is that the AM microbial inoculum is suspension, dark brown, tool alcohol tart flavour, organic acid total amount 1.56%, total amino acid content 380.2mg/ml also has materials such as VITAMIN and physiologically active.
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| CN106954418A (en) * | 2017-04-14 | 2017-07-18 | 山东永春堂集团有限公司 | A kind of application method for the liquid microbe bacterial manure for promoting acer truncatum to grow |
| CN107974423A (en) * | 2017-12-25 | 2018-05-01 | 广东杰士农业科技有限公司 | A kind of biological soil activating agent and preparation method thereof |
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