CN103184174A - Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium - Google Patents
Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium Download PDFInfo
- Publication number
- CN103184174A CN103184174A CN201110455742XA CN201110455742A CN103184174A CN 103184174 A CN103184174 A CN 103184174A CN 201110455742X A CN201110455742X A CN 201110455742XA CN 201110455742 A CN201110455742 A CN 201110455742A CN 103184174 A CN103184174 A CN 103184174A
- Authority
- CN
- China
- Prior art keywords
- subtilis
- culture
- solid
- seed
- fermentation material
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention relates to a microbe biological agent, and concretely relates to a production method of a bacillus subtilis biological agent used for a sodium humate-containing feed in a medium. The bacillus subtilis bacterial strain is subjected to first-stage solid seed culture and second-stage liquid seed fermentation culture to obtain a bacterial classification liquid. The bacterial classification liquid is inoculated in the medium which takes glucose, sodium humate, soybean meal, corn and wheat bran as raw materials for three-stage solid aerobic fermentation, the culture condition is adjusted to form spore, the spore can be treated, dried, and crushed to produce the bacillus subtilis biological agent. The biological agent contains more than or equal to 200 hundreds million/g of acillus subtilis, the pH value is 5.6-6.6, the moisture is less than or equal to 10%, and the granularity is greater than or equal to 100 meshes, the color presents special shallow grey or grey black, and the shelf life is 18 months. The biological agent can be taken as an additive application in aquatic product, and livestock and poultry culture, can increase the production performance and improve the culture environment.
Description
Technical field
The present invention relates to the microorganism biological preparation, the feed production method of high density subtilis biotechnological formulation that contains Sodium salts humic acids specifically in a kind of substratum.
Background technology
Subtilis (Bacillus subtilis) is a kind of of bacillus, 0.7～0.8 * 2～3 microns of individual cells, and gram-positive microorganism, no pod membrane, peritrichous can move.Gemma is big, and is oval to column, and 0.6～0.9 * 1.0～1.5 microns, be positioned at thalline central authorities or inclined to one side slightly, gemma forms the back thalline and does not expand.The bacterium colony surface irregularity, dirty white or little yellow, when liquid culture, the normal wrinkle mould that forms.Characteristics such as subtilis has that reproduction speed is fast, high temperature resistant, acid and alkali-resistance, anti-extruding, stability are good, applied range, Chang Zuowei type strain in the scientific research of modern molecular genetics is explored is widely used in all many-sides such as foodstuffs industry, fodder industry, aquaculture, environment protection and health care medicine in the production practice of reality; Therefore, more and more be subjected to scientific worker and business people's attention.
Contain biologically active substances such as rich in protein, amino acid, organic acid, VITAMIN, unsaturated fatty acids, proteolytic enzyme, amylase, lipase and peptide antibiotics in subtilis thalline and the secretory product thereof.Therefore, higher using value is all arranged in livestock and poultry cultivation and aquaculture.
In former studies, the scientific worker all has further investigation to the aspects such as taxonomy status, physiological and biochemical property, fermentation condition and regulation and control thereof of subtilis.In recent years, the subtilis applied research is also obtained very big progress.But with high density fermentation of bacillus subtilis technology report and few, the feed that substratum is contained Sodium salts humic acids is not with the development of high density subtilis biotechnological formulation and production and appear in the newspapers to feed.
The present invention adopts bacillus subtilis strain, through one-level solid seed culture, liquid secondary seed fermentation culture, the solid medium that employing contains Sodium salts humic acids carries out aerobic fermentation, go out the gemma processing through adjusting culture condition again, dry, pulverizing are produced substratum and are contained Sodium salts humic acids, feed high density subtilis biotechnological formulation product.This technological design and production still belong to internal and international initiative.
Sodium salts humic acids is larger molecular organics matter, mainly is hydroxyl, phenolic hydroxyl group, methoxyl group, quinonyl etc.Comprehensive every report, humic acids are applied in the animal and fowl fodder and can adsorb enteron aisle inflammatory thing and toxic substance, and intestinal mucosa is shielded; Can suppress and kill pathogen enterobacteria, improve the utilization ratio of feed, and the hemostasis and pain-relieving effect is arranged; Ammonia in the adsorbable ight soil, hydrogen sulfide alleviate the stink of ight soil; The Sodium salts humic acids resource is extensive, cheap, drug residue free, without any incompatibility, is beneficial to environmental protection.But the active group of humic acids is often sealed by impurity, almost can not play a role, and can improve its biologic activity greatly by fermentative processing.Sodium salts humic acids is used as the sporeformer preparation media, will promote the preparation biopotency.
Product function of the present invention is: 1. utilize the strong characteristics of sporeformer enzymatic productivity, improve efficiency of feed utilization; 2. after sporeformer enters stomach and intestine, through recovery propagation, can consume enteron aisle oxygen, be conducive to anaerobism probiotic bacteriums such as lactic acid, bifid and play a role; 3. Sodium salts humic acids is behind microbial fermentation, and biological activity strengthens, and effect has synergy to sporeformer; 4. sporeformer enters behind the hindgut even gets rid of and can also play a role after external, can significantly improve breeding environment.
The feed that contains Sodium salts humic acids in the substratum is nontoxic, harmless, without any side effects with high density subtilis biotechnological formulation product, is a kind of microbial preparation additive of green, has vast market prospect.
At present, in livestock and poultry cultivation and aquaculture, improve protein utilization, reducing the microbiotic use is urgent problem.And the present invention exactly helps to solve above-mentioned two problems or a difficult problem very much.Product in Guangdong, regional widespread use such as Hubei, Jiangsu, Hebei, Shandong, Beijing, Shenyang, Dalian, obtained good economic benefits and social benefit.
Summary of the invention
The object of the invention is to provide the feed that contains Sodium salts humic acids in a kind of substratum production method of high density subtilis biotechnological formulation.
For achieving the above object, the technical solution used in the present invention is: be bacterial strain with the subtilis, through one-level solid seed culture, the subtilis seed liquor is produced in the fermentation of secondary liquid seeds.Be main medium with glucose, Sodium salts humic acids, dregs of beans, corn, wheat bran etc., seed liquor is inoculated in carries out three grades of solid aerobic fermentations on the substratum, adjust culture condition afterwards and go out the gemma processing, drying is again pulverized, and produces to contain subtilis (cfu) 〉=20,000,000,000/g, pH value 5.6～6.6, moisture≤10%, granularity 〉=100 orders, color and luster presents distinctive light gray or grey black, the biotechnological formulation product of 18 months quality guaranteed perioves.
Contain the feed of Sodium salts humic acids in a kind of substratum with the production method of high density subtilis biotechnological formulation, comprise the steps:
1) single bacterium colony purification of seed is with preferred: with the subtilis slant strains, be coated with at the culture dish that fills bacteria culture medium, through cultivating, obtain the single bacterium colony seed of subtilis;
2) one-level solid seed culture: the single bacterium colony seed that obtains in the step 1) is inoculated into the 200ml eggplant-shape bottle, through cultivating, obtains one-level solid seed;
3) the eggplant-shape bottle seed that obtains liquid fermentation and culture secondary seed: with step 2) is inoculated in the fermentor tank liquid nutrient medium, and the inoculative proportion of eggplant-shape bottle and liquid nutrient medium is 1 bottle: 200 kilograms.Obtain the secondary liquid seeds through cultivation.The fermentation liquid seed standard that obtains is: color is normal, and free from extraneous odour, gemma account for total count more than 20%, pH 6.0～6.5, the thalline size is 0.7～0.8 * 2～3 μ m, and gemma 0.6～0.9 * 1.0～1.5 μ m contain subtilis (cfu) 〉=20,000,000,000/ml.
4) three grades of solid fermentation material aerobic fermentations: the secondary liquid seeds that obtains in the step 3) is inoculated in the solid fermentation material, and through the ventilation oxygen supply, fermentation culture obtains the solid fermentation material through cultivating.
5) going out gemma handles: the solid fermentation material that obtains in the step 4) is gone out the brood cell handles, namely stop the oxygen supply of ventilating, with its spread out, overturning, reduce temperature and humidity, impel the subtilis that contains in the solid fermentation material to form gemma.
6) drying and pulverization process: the formation that step 5) is obtained brood cell's solid fermentation material dry processing, by pulverizing, produce the feed usefulness high density subtilis biotechnological formulation product that contains the Sodium salts humic acids that ferment that contains subtilis (cfu) 〉=20,000,000,000/g.
In the above-mentioned subtilis culturing process, step 1) and 2) weight of the subtilis solid seed culture medium that adopts consists of: glucose 0.8～1.2%, peptone 0.8～1.2%, extractum carnis 0.3～0.6%, sodium-chlor 0.03～0.07%, sal epsom 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, surplus are pure water, pH6.8～7.2, agar 1.5～2.2%;
Sterilising conditions: 0.08MPa, 115 ℃, 18min;
Culture temperature: 28 ℃～32 ℃, the time is 48～72h.
In the above-mentioned subtilis culturing process, the weight of the subtilis seed fermentation substratum that step 3) adopts consists of: glucose 0.5～1.5%, peptone 0.2～0.8%, yeast powder 0.2～0.8%, Semen Maydis powder 1.0～3.0%, dregs of beans 1.0～3.0%, ammonium sulfate 0.1～0.3%, sodium-chlor 0.03～0.07%, sal epsom 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, surplus are pure water, pH6.8～7.2;
Sterilising conditions: 0.1MPa, 121 ℃, 20min;
Fermentation culture conditions: tank pressure 0.01～0.10Mpa, air flow 0.5～1.0V/Vmin, rotating speed 100-200rpm, 28 ℃～34 ℃ of temperature, time are 48～72h;
Above-mentioned steps 4) in the described solid fermentation material aerobic fermentation process, various raw material weight ratios are in the solid fermentation material: 1～3 part of glucose, 10～40 parts of Sodium salts humic acidss, 10～20 parts of dregs of beans, 10～20 parts of Semen Maydis powders, 50～70 parts in wheat bran; The liquid secondary seed is respectively with the part by weight that the solid fermentation material mixes: 1. 30: 100,2. 40: 100,3. 50: 100; Fermentation culture conditions: air flow 0.5～1.0V/Vmin, 50 ℃～60 ℃ of leavening temperatures, fermentation time 48～64h.
Step 5) is described to be gone out during gemma handles: need stop the oxygen supply of ventilating to the solid fermentation material through cultivating, with its spread out, overturning.
Go out the gemma treatment condition: 20 ℃～30 ℃ of temperature, water content 20～30%, time 24～48h;
Step 6) is carried out drying treatment and pulverizing with the described brood cell's of containing of step 5) solid fermentation material, and the drying treatment condition is: adopt drying oven to carry out drying, 60 ℃～80 ℃ of temperature, 38 ℃～42 ℃ of drying oven temperature outs, time 8～10min in the drying oven;
The pulverization process condition is: carry out under aseptic cleaning condition, pulverize back granularity 80～120 orders, water content 8～15%, contain 200～22,000,000,000/g of subtilis (cfu).
Preferred version is as follows:
The weight of the subtilis seed culture medium that adopts consists of: glucose 1.0%, peptone 1.0%, extractum carnis 0.5.%, sodium-chlor 0.05%, sal epsom 0.05%, dipotassium hydrogen phosphate 0.1%, surplus are pure water, pH7.0, agar 2.0%.
Sterilising conditions: 0.08MPa, 115 ℃, 18min;
Culture temperature: 30 ℃, the time is 48h.
The weight of the subtilis secondary seed fermention medium that adopts consists of: glucose 1.0%, peptone 0.5%, yeast powder 0.5%, Semen Maydis powder 2.0%, dregs of beans 2.0%, ammonium sulfate 0.2kg%, sodium-chlor 0.05%, sal epsom 0.05%, dipotassium hydrogen phosphate 0.10%, surplus are pure water, pH7.0.
Sterilising conditions: 0.1MPa, 121 ℃, 20min;
Fermentation culture conditions: tank pressure 0.05Mpa, air flow 0.5V/Vmin, rotating speed 100rpm, 30 ℃ of culture temperature, time are 48h.
In the solid fermentation material aerobic fermentation process that adopts, various raw material weight ratios are in the solid fermentation material: 50 parts of 2 parts of glucose, 30 parts of Sodium salts humic acidss, 10 parts of dregs of beans, Semen Maydis powder 10, wheat bran; The part by weight that liquid seeds mixes with the solid fermentation material 40: 100.Fermentation culture conditions is: air flow 0.5V/Vmin, 50 ℃ of leavening temperatures, fermentation time 48.
The solid fermentation material that adopts goes out during gemma handles, need stop the oxygen supply of ventilating to the solid fermentation material through cultivating, with its spread out, overturning.
Going out the gemma treatment condition is: 20 ℃ of temperature, water content 20, time 48h;
During the described brood cell's of containing solid fermentation material carries out drying treatment and pulverizes, adopt drying oven to carry out
Drying, the drying treatment condition is: 60 ℃ of temperature, drying oven temperature out in the drying oven: 38 ℃, time 8min;
The pulverization process condition is: carry out under aseptic cleaning condition, pulverize back granularity 100 orders, water content 10%, contain 20,000,000,000/g of subtilis (cfu).
Use the colony counting method inspection, the feed that contains the Sodium salts humic acids that ferments in the substratum is with high density subtilis biotechnological formulation product, contains subtilis (cfu) 〉=20,000,000,000/g.
Contain the feed of Sodium salts humic acids in the described substratum with high density subtilis biotechnological formulation product, can be used as the high-quality biological feed additive in aquatic products, the livestock and poultry cultivation, the water quality cleansing agent that also can be used as seawater, fresh water is used.
The present invention has following advantage:
The present invention has that bacterial classification is selected suitably, technology is new, bacterium is counted height, product is many, wide adaptability, reliable for effect, be of value to the characteristics that environment etc. is given prominence to.
1. bacterial classification is selected suitably, reproduction speed is fast, product is many.Bacillus subtilis bacterial strain source is very extensive, all extensively exist on land and ocean, and has self outstanding characteristic, 1) reproduction speed is fast: the growth Dai Shiwei of subtilis 18 minutes, just can reach logarithmic phase in 2 hours during the fermentation, 24 hours just can arrive bacterium number breeding peak, and fermentation in 48 hours reaches terminal point, adjust carbon-nitrogen ratio, bacterium number (cfu) can reach 20,000,000,000/more than the ml.2) nutritious, can secrete and enzyme products such as proteolytic enzyme, amylase and cellulase that metabolism is a large amount of, be rich in each seed amino acid, organic acid and antibacterial peptide.3) strong stress resistance: high temperature resistant, become gemma after, can tolerate the high temperature more than 60 ℃ for a long time, restrained oneself 80 degree 20 minutes, or even moment 100 ℃ of high temperature, but survival rate more than 90% still.Acid and alkali-resistance can adapt to the soda acid scope of pH 2～9.Produce in the extruding particle process in general feeds, survival rate remains on more than 90%.4) stability is good, anti-standing storage, and under various site of deployment conditions, survival is not affected substantially.Under room temperature, sealing, lucifuge condition, can guarantee the quality more than 18 months.
2. new, the good product quality of technology.The present invention produces bacterial strain, through one-level solid seed culture, the fermentation of secondary liquid seeds, three grades of solid fermentation material aerobic fermentations, go out the gemma processing through adjusting culture condition again, the feed that contains Sodium salts humic acids in substratum high density subtilis biotechnological formulation product is produced in dry, pulverizing, and production technique still belongs to internal and international initiative.The technical measures that relate to by this project can guarantee to produce the feed that contains the Sodium salts humic acids that ferments in the substratum with high density subtilis biotechnological formulation, and make the gemma number (cfu) of product reach 20,000,000,000/more than the g.Production starting material of the present invention are formed simple, wide material sources, and are cheap, and to adopt Sodium salts humic acids be nutrient media components, have the particular appearance feature and can strengthen effect.
3. wide adaptability, effect are reliable.The product that the present invention produces can be applicable to all kinds of fodder production such as complete diet pellet, concentrate feed, Preblend, also can directly add in plant and feed, and is widely used in aquaculture and livestock and poultry cultivation.A large amount of production practice prove: in holothruian cultures, use, can strengthening immunity and disease resistance, and reduce ammonia-nitrogen content in the water body, purify water, prolong 5～7 days adherance time of changing down, raising sea cucumber output 10.9%.In egg fowl is cultured, use, can strengthen poult immunizing power, reduce death rate 1.8%, improve laying rate 2.02%.In piglet and growing and fattening pigs breed, use, can prevent and treat yellow and white dysentery of piglet, improve the growing and fattening pigs speed of weight increment, reduce the microbiotic usage quantity.
4. improve breeding environment, be of value to ecological.Subtilis can extensively utilize carbon source and nitrogenous source, in the breed of sea cucumber, can utilize water body small molecules nitrogenous compound, remaining nitrogenous bait and sea cucumber ight soil etc., can promote to culture the degraded of organism nitrogenous compound at the bottom of the pond, ammonia nitrogen, hydrogen sulfide in the aquaculture pond water body are descended, make item indexs such as COD, BOD value and pH value reach normal, purify water, improve dissolved oxygen, improve breeding environment, improve sea cucumber output.In livestock and poultry cultivation, use, can alleviate the ammonia odor of ight soil in the breeding environment, can obviously improve breeding environment, be of value to ecological.
5. the feed that contains the Sodium salts humic acids that ferments in the substratum is with high density subtilis biotechnological formulation product, and nutrition is complete, have no side effect, and security is good.In feed, use, but strengthening immunity improves surviving rate, promotes to grow, and promotes the aquatic animal quality.
Embodiment
The single bacterium colony of subtilis (Bacillus subtilis) is carried out purifying and cultivates, goes out the gemma processing through the cultivation of solid first order seed, the cultivation of liquid second order fermentation, three grades of solid fermentation material aerobic fermentations, processes such as dry and pulverizings can be produced and contain the feed usefulness high density subtilis biotechnological formulation product that contains the Sodium salts humic acids that ferments in the substratum.The concrete operations step is as follows:
Embodiment 1:
1) single bacterium colony purifying is with preferred: with subtilis inclined-plane bacterial strain, be coated with at the culture dish that fills bacteria culture medium, through cultivating, obtain the single bacterium colony seed of subtilis.
2) one-level solid seed culture: with the single bacterium colony seed that obtains in the step 1), choose single colony inoculation in one-level solid seed culture medium, solid seed culture bottle is the 200ml eggplant-shape bottle, and loading amount is the 50ml solid medium, through cultivating, obtains one-level solid seed;
3) single bacterium colony purifying and one-level solid seed culture medium is preferred
Under laboratory condition, cultivate the optimization test of the factor, test design is 6 factors, 3 levels, test design and test-results such as following table 1:
Table 1. subtilis one-level solid seed culture medium formula test
Determine that from table 1 test-results single bacterium colony purifying and solid seed culture medium are: glucose 10g, peptone 10g, extractum carnis 4g, sodium-chlor 0.5g, sal epsom 0.5g, dipotassium hydrogen phosphate 1g, 1000ml pure water, pH7.0, agar 20g; Culture temperature is 30 ℃, and the time is 64h.
Embodiment 2:
1) secondary liquid seeds fermentation culture: take tank fermentation method to produce liquid seeds, cultured one-level solid seed is received in the seed fermentation jar, one-level solid seed and 1 ton of fermentor tank (liquid amount 70%) inoculative proportion are 1 bottle: 200 kilograms.Inoculation is cultivated by aerobic fermentation, and when cultivating maturation, bacterium liquid has a kind of little sour fragrance, test under microscope subtilis thalline stalwartness, and unusual phenomenoies such as no living contaminants namely can be used as the secondary liquid seeds and use for the solid fermentation material.
2) secondary liquid seeds fermention medium optimization test
Under the fermentation plant condition, carry out the optimization test that fermentation tank culture medium is cultivated, test design is 6 factors, 3 levels, test design and test-results such as following table 2:
Table 2. subtilis secondary liquid seed culture medium formula test
According to table 2 test-results, determine that secondary liquid seeds fermention medium is: glucose 10g, peptone 5.0g, yeast powder 5.0g, Semen Maydis powder 20%, dregs of beans 2.0%, sodium-chlor 0.5g, sal epsom 0.5g, dipotassium hydrogen phosphate 1.0g, 1000ml pure water, pH7.0.
3) secondary liquid seeds fermentation culture conditions test
Adopt above-mentioned fermention medium, under the fermentation plant condition, carry out the ferment tank condition test.Design 5 factors, 3 levels, test design and test-results such as following table 3:
The test of table 3. subtilis liquid seeds jar fermentation culture conditions
According to experimental result, fermentation culture conditions is defined as: tank pressure 0.05Mpa, air flow 0.5V/Vmin, rotating speed 100rpm, and culture temperature is 30 ℃, the time is 48h.
4) subtilis microscopy: bacillus subtilis mycetocyte size is about 0.7～0.8 * 2～3 microns, uniform coloring.No pod membrane, peritrichous can move.Gram-positive microorganism, 0.6～0.9 * 1.0～1.5 microns of gemma, oval to column, be positioned at thalline central authorities or inclined to one side slightly, gemma forms the back thalline and does not expand.
5) the subtilis culture dish checks: the subtilis bacterium colony surface irregularity on the plate culture medium is opaque, dirty white or little yellow, and the edge often forms fold, and liquid culture often forms the wrinkle mould.
Embodiment 3:
1) three grades of solid fermentation material fermentation culture: liquid seeds is inoculated in the solid fermentation material, and through the ventilation oxygen supply, fermentation culture obtains the solid fermentation material through cultivating.
2) various raw material weight ratios in the solid fermentation material: 50 parts of 2 parts of glucose, 30 parts of Sodium salts humic acidss, 10 parts of dregs of beans, Semen Maydis powder 10, wheat bran; The part by weight that the secondary liquid seeds mixes with the solid fermentation material is 40: 100.Through the ventilation oxygen supply, air flow 0.5V/Vmin.The solid fermentation material of different moisture content sees the following form to Temperature Influence:
Table 4. different moisture content solid fermentation material to the fermentation of bacillus Temperature Influence (℃)
Under the situation of above-mentioned solid fermentation material formula, table 4 test-results shows: the relative water content ratio that the solid fermentation material mixes is 40: 100, between 55 ℃～60 ℃ of the leavening temperatures, can keep fermentation time 48h 24 hours.
3) inspection of bacterium number and definite: with the step 4) and 5 in above-described embodiment 3) identical.Institute's difference is that the solid fermentation material is had a dilution.Elder generation's five point sampling methods, each point sampling amount 100g mixes back taking-up 1g and is dissolved in the 100ml sterilized water, and normal temperature shook 20 minutes.
4) going out gemma handles: temperature and time sees Table 5 to the influence that the solid fermentation material goes out gemma.
Table 5. temperature and time goes out the influence of gemma to the solid fermentation material
Table 5 is the result show: going out gemma processing top condition is: 20 ℃ of temperature, water content 20, time 48h; With this understanding, have more than 90% and can both become gemma.
5) drying and pulverization process: the influence to gemma of kiln temperatures and time sees Table 6.
Table 6. differing temps different time drying oven is to the influence of gemma
Table 6 is the result show: drying oven differing temps and time have influence to gemma.Guaranteeing that top condition is 60 ℃ of temperature, 38 ℃ of drying oven temperature outs, time 8min in the drying oven under the product water content conformance with standard requirement situation.
The pulverization process condition: under aseptic cleaning condition, carry out, pulverize back granularity 100 orders, water content 10%, contain subtilis (cfu) 20,000,000,000/more than the g, be qualified product.
Embodiment 4:
Contain the feed of Sodium salts humic acids in the substratum that embodiment 3 is obtained with the product of high density subtilis, add by 0.05～0.1wt% at the daily ration of piglet and carry out feeding experiment, the results are shown in Table 7:
Table 7. is used the feed that contains Sodium salts humic acids and is used high density producing bacillus subtilis product to the influence of piglet day weight gain and material anharmonic ratio
By table 7 as can be seen: add the product that the feed that contains Sodium salts humic acids in 0.05～0.1% substratum is used the high density subtilis in the daily ration of piglet, test group is than control group, and day weight gain obviously increases, and diarrhea disease percentage obviously descends.
Embodiment 5
Contain the feed of Sodium salts humic acids in the substratum with embodiment 3 acquisitions with high density producing bacillus subtilis product, in sea blue brown shell layer chicken daily ration, add 0.05wt% and carry out feeding experiment, the results are shown in Table 8:
The feed that contains Sodium salts humic acids in table 8 substratum produces Effect on Performance with high density producing bacillus subtilis product to sea blue brown laying hen chicken all living creatures
As can be seen from Table 8: at 287 sea blue brown shell layer chickens, through 35 day trial period, test group is not than control group (add in the daily ration feed that contains Sodium salts humic acids in the substratum that embodiment 3 obtains with high density producing bacillus subtilis product), and laying rate improves 2.02%, and day per unit area yield improves 1.44g/, death rate reduces by 1.8%, feedstuff-egg ratio reduces by 6.0%, and total egg productivity improves 15.4kg, and eggshell hardness is strong, gloss is bright good, the color even unanimity; The chicken manure moulding, stink, ammonia odor alleviate, and breeding environment obviously improves.
Embodiment 6
The product that contains the usefulness high density subtilis of Sodium salts humic acids feed in the substratum with embodiment 3 acquisitions, join to raise in the water body the children and carry out application test, the feed that contains Sodium salts humic acids in the substratum sees Table 9 with high density producing bacillus subtilis product to the influence that sea cucumber increases weight:
The feed that contains Sodium salts humic acids in table 9. substratum uses high density producing bacillus subtilis product to the influence of sea cucumber weightening finish
* test period: on March 30,15 days～2011 March in 2011
* test temperature: 16 ℃ of sea cucumber pond water temperatures;
* test volume: each cultures pond is the 15m3 water body; Each water body refers to length * wide * height=1m * 1m * 1m=1m3 (cubic meter water body)
* 1 every day of test group each water body with 5ml feed with high density producing bacillus subtilis product;
* 2 every days of test group each water body with 10ml feed with high density producing bacillus subtilis product;
* control group each water body every day 5ml seawater;
By table 9 as can be seen, when 16 ℃ of water temperatures, through 15 days nursing, test group 1 on average increased production 11.0% than control group; Test group 2 on average increases production 13.1% than control group.Test-results shows that it is obvious with high density producing bacillus subtilis product effect to add the feed that contains Sodium salts humic acids in the substratum in the holothruian cultures.
Embodiment 7
Process by embodiment 1-6 is operated, be with embodiment 1-6 difference, for improving the quality of products, shorten the production cycle, the high-activity microorganism bacterial strain that the applicant has also tried out Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences's separation, purifying and identified, as highly active subtilis, yeast saccharomyces cerevisiae, Lactobacterium acidophilum and photosynthetic bacterium, and attempt Application of composite, effect is better.
The present invention is bacterial strain with the subtilis, be main production raw material with glucose, Sodium salts humic acids, dregs of beans, corn, wheat bran etc., carrying out aerobic fermentation under 30 ℃～32 ℃ conditions cultivates, changing culture condition afterwards promotes gemma to form, produce and contain subtilis (cfu) 〉=20,000,000,000/g, pH value 5.6～6.6, moisture≤10%, granularity 〉=100 orders, color and luster present the feed that contains Sodium salts humic acids in distinctive light gray or grey black, the substratum of 18 months quality guaranteed perioves with high density subtilis biotechnological formulation product.Production technique of the present invention is simple, easy handling; Raw materials used wide material sources, low being easy to of cost promoted.In livestock and poultry and aquaculture, use, can improve immunizing power and surviving rate, promote to grow and, improve breeding performonce fo animals, can purify water and improve breeding environment, be a kind of desirable high-quality microorganism feed addictive.
Claims (9)
1. contain the Sodium salts humic acids feed with the production method of subtilis biotechnological formulation in a substratum, it is characterized in that, comprise the steps:
1) single bacterium colony purification of seed is with preferred: with the subtilis slant strains, be coated with at the culture dish that bacteria culture medium is housed, through cultivating, obtain the single bacterium colony seed of subtilis;
2) one-level solid seed culture: with the single bacterium colony seed that obtains in the step 1), choose single colony inoculation in one-level solid seed culture medium, solid seed culture bottle is the 200ml eggplant-shape bottle, and loading amount is solid medium 50ml, through cultivating, obtains one-level solid seed;
3) the one-level solid seed (eggplant-shape bottle bacterial classification) that obtains secondary liquid seeds fermentation culture: with step 2) is inoculated in the second order fermentation jar liquid nutrient medium, the inoculum size of one-level solid seed and second order fermentation jar liquid seeds nutrient solution is: one-level solid seed and 1 ton of fermentor tank (liquid amount 70%) inoculative proportion are 1 bottle: 200 kilograms, through cultivating, obtain the secondary liquid seeds;
4) three grades of solid fermentation material aerobic fermentations: the secondary liquid seeds that obtains in the step 3) is inoculated in the solid fermentation material, and the part by weight that the secondary liquid seeds mixes with the solid fermentation material is 40: 100; Through the ventilation oxygen supply, fermentation culture obtains the solid fermentation material through cultivating;
5) going out gemma handles: the solid fermentation material that obtains in the step 4) is gone out the brood cell handle, namely to the solid fermentation material through cultivating, stop the oxygen supply of ventilating, with its spread out, overturning, temperature is reduced to 20 ℃～30 ℃, water content is reduced to 20%～30%, impels the subtilis that contains in the solid fermentation material to form gemma;
6) drying and pulverization process: the solid fermentation material that contains the brood cell that obtains in the step 5) is carried out drying treatment, the oven dry of drying stove, pulverizing mill is pulverized, and the feed of producing subtilis (cfu) 〉=20,000,000,000/g that contains Sodium salts humic acids in the substratum is with high density subtilis biotechnological formulation product.
2. according to the described production method of claim 1, it is characterized in that: the weight of the subtilis solid seed culture medium that step 1) and 2) adopts consists of: glucose 0.8～1.2%, peptone 0.8～1.2%, extractum carnis 0.3～0.6%, sodium-chlor 0.03～0.07%, sal epsom 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, surplus are pure water, pH6.6～7.2, agar 1.5～2.2%;
Medium sterilization condition: 0.08MPa, 115 ℃, 18min;
Culture temperature: 28 ℃～32 ℃, the time is 48～72h.
3. according to the described cultural method of claim 2, it is characterized in that: the weight of the subtilis solid seed culture medium that adopts consists of: glucose 1.0%, peptone 1.0%, extractum carnis 0.5.%, sodium-chlor 0.05%, sal epsom 0.05%, dipotassium hydrogen phosphate 0.1%, surplus are pure water, pH7.0, agar 2.0%;
Medium sterilization condition: 0.08MPa, 115 ℃, 18min;
Culture temperature: 30 ℃, the time is 48h.
4. according to the described cultural method of claim 1, it is characterized in that: the weight of the subtilis seed fermentation substratum that step 3) adopts consists of: glucose 0.5～1.5%, peptone 0.2～0.8%, yeast powder 0.2～0.8%, Semen Maydis powder 1.0～3.0%, dregs of beans 1.0～3.0%, ammonium sulfate 0.1～0.3%, sodium-chlor 0.03～0.07%, sal epsom 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, surplus are pure water, pH6.6～7.2;
Sterilising conditions: 0.1MPa, 121 ℃, 20min;
Fermentation culture conditions: tank pressure 0.01～0.10Mpa, air flow 0.5～1.0V/Vmin, rotating speed 100～200rpm, 28 ℃～34 ℃ of temperature, time are 48～72h.
5. according to the described cultural method of claim 4, it is characterized in that: the weight of the subtilis seed fermentation substratum that adopts consists of: glucose 1.0%, peptone 0.5%, yeast powder 0.5%, Semen Maydis powder 2.0%, dregs of beans 2.0%, ammonium sulfate 0.2kg%, sodium-chlor 0.05%, sal epsom 0.05%, dipotassium hydrogen phosphate 0.10%, surplus are pure water, pH7.0;
Sterilising conditions: 0.1MPa, 121 ℃, 20min;
Fermentation culture conditions: tank pressure 0.05Mpa, air flow 0.5V/Vmin, rotating speed 100rpm, 30 ℃ of culture temperature, time are 48h;
The fermented liq secondary seed standard that obtains is: color is normal, and free from extraneous odour, gemma account for total count more than 20%, pH is 6.0～6.5, the thalline size is 0.7～0.8 * 2～3 μ m, and gemma 0.6～0.9 * 1.0～1.5 μ m contain subtilis (cfu) 〉=20,000,000,000/ml.
6. according to the described cultural method of claim 1, it is characterized in that: in the described solid fermentation material aerobic fermentation process, various raw material weight ratios are in the solid fermentation material: 1～3 part of glucose, 10～40 parts of Sodium salts humic acidss, 10～20 parts of dregs of beans, 10～20 parts of Semen Maydis powders, 50～70 parts in wheat bran;
Solid fermentation material culture condition: air flow 0.5～1.0V/Vmin, 50 ℃～60 ℃ of leavening temperatures, fermentation time 48～64h.
7. according to the described cultural method of claim 6, it is characterized in that: in the described solid fermentation material aerobic fermentation process, various raw material weight ratios are in the solid fermentation material: 50 parts of 2 parts of glucose, 30 parts of Sodium salts humic acidss, 10 parts of dregs of beans, Semen Maydis powder 10, wheat bran;
Solid fermentation material culture condition: air flow 0.5V/Vmin, 50 ℃ of leavening temperatures, fermentation time 48.
8. according to the described cultural method of claim 6, it is characterized in that: step 5) is described to be gone out during gemma handles: need stop the oxygen supply of ventilating to the solid fermentation material through cultivating, with its spread out, overturning; Going out the gemma treatment condition is: 20 ℃～30 ℃ of temperature, water content 20～30%, time 24～48h;
Step 6) is carried out drying treatment and pulverizing with the described brood cell's of containing of step 5) solid fermentation material, and the drying treatment condition is: adopt drying oven to carry out drying, 60 ℃～80 ℃ of temperature, 38 ℃～42 ℃ of drying oven temperature outs, time 8～10min in the drying oven;
The pulverization process condition is: carry out under aseptic cleaning condition, pulverize back granularity 80～120 orders, contain 200～22,000,000,000/g of subtilis (cfu), water content 8～15%.
9. according to the described cultural method of claim 8, it is characterized in that: describedly go out during gemma handles: need stop the oxygen supply of ventilating to the solid fermentation material through cultivating, with its spread out, overturning; Going out the gemma treatment condition is: 20 ℃ of temperature, water content 20, time 48h;
The described brood cell's of containing solid fermentation material carries out drying treatment and pulverizing, and the drying treatment condition is: adopt drying oven to carry out drying, 60 ℃ of temperature, 38 ℃ of drying oven temperature outs, time 8min in the drying oven;
The pulverization process condition is: carry out under aseptic cleaning condition, pulverize back granularity 100 orders, water content 10%, contain 20,000,000,000/g of subtilis (cfu).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110455742.XA CN103184174B (en) | 2011-12-30 | 2011-12-30 | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110455742.XA CN103184174B (en) | 2011-12-30 | 2011-12-30 | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103184174A true CN103184174A (en) | 2013-07-03 |
| CN103184174B CN103184174B (en) | 2014-11-19 |
Family
ID=48675625
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110455742.XA Active CN103184174B (en) | 2011-12-30 | 2011-12-30 | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103184174B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103652326A (en) * | 2013-10-16 | 2014-03-26 | 内蒙古斯隆生物技术有限责任公司 | Polypeptide protein feed containing humic acid and preparation method thereof |
| CN104651246A (en) * | 2013-11-20 | 2015-05-27 | 丰益(上海)生物技术研发中心有限公司 | Bacillus subtilis and soybean meal fermentation pre-treatment process |
| CN105028906A (en) * | 2015-08-20 | 2015-11-11 | 岳先合 | Special raising feed for Chinese sturgeon and preparation method thereof |
| CN105062931A (en) * | 2015-09-07 | 2015-11-18 | 山东寰达生物科技有限公司 | Preparation method of high-concentration bacillus subtilis Cohn and application of high-concentration bacillus subtilisCohn in aquaculture |
| CN105861615A (en) * | 2016-04-22 | 2016-08-17 | 闫大凤 | Production method of amino acid bioactive peptide for aquatic products |
| CN106107023A (en) * | 2016-08-26 | 2016-11-16 | 北京斯隆生物技术有限责任公司 | A kind of microbial degradation humic acids feedstuff and preparation method thereof |
| CN108651758A (en) * | 2018-07-25 | 2018-10-16 | 石阡县毅峰农牧开发有限公司 | One kind chicken feed containing phytase and preparation method thereof |
| CN111607627A (en) * | 2020-05-12 | 2020-09-01 | 南京大方生物工程有限公司 | Fermentation process for producing antibacterial peptide bacillus subtilis and application of fermentation process in disease-resistant and yield-increasing of livestock and poultry |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168045A (en) * | 2010-12-24 | 2011-08-31 | 北京科为博生物科技有限公司 | Bacillus subtilis preparation and preparation method thereof |
-
2011
- 2011-12-30 CN CN201110455742.XA patent/CN103184174B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168045A (en) * | 2010-12-24 | 2011-08-31 | 北京科为博生物科技有限公司 | Bacillus subtilis preparation and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 潘军 等: "腐殖酸钠发酵饲料的研制及其在仔猪育肥中的应用效果", 《第八届全国绿色环保肥料（农药）新技术、新产品交流会论文集》 * |
| 郑寿亭: "《微生物酶及其应用》", 30 April 1973, 山东人民出版社 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103652326A (en) * | 2013-10-16 | 2014-03-26 | 内蒙古斯隆生物技术有限责任公司 | Polypeptide protein feed containing humic acid and preparation method thereof |
| CN103652326B (en) * | 2013-10-16 | 2016-07-06 | 内蒙古斯隆生物技术有限责任公司 | A kind of polypeptide protein feed containing humic acids and preparation method thereof |
| CN104651246A (en) * | 2013-11-20 | 2015-05-27 | 丰益(上海)生物技术研发中心有限公司 | Bacillus subtilis and soybean meal fermentation pre-treatment process |
| CN105028906A (en) * | 2015-08-20 | 2015-11-11 | 岳先合 | Special raising feed for Chinese sturgeon and preparation method thereof |
| CN105062931A (en) * | 2015-09-07 | 2015-11-18 | 山东寰达生物科技有限公司 | Preparation method of high-concentration bacillus subtilis Cohn and application of high-concentration bacillus subtilisCohn in aquaculture |
| CN105861615A (en) * | 2016-04-22 | 2016-08-17 | 闫大凤 | Production method of amino acid bioactive peptide for aquatic products |
| CN106107023A (en) * | 2016-08-26 | 2016-11-16 | 北京斯隆生物技术有限责任公司 | A kind of microbial degradation humic acids feedstuff and preparation method thereof |
| CN108651758A (en) * | 2018-07-25 | 2018-10-16 | 石阡县毅峰农牧开发有限公司 | One kind chicken feed containing phytase and preparation method thereof |
| CN111607627A (en) * | 2020-05-12 | 2020-09-01 | 南京大方生物工程有限公司 | Fermentation process for producing antibacterial peptide bacillus subtilis and application of fermentation process in disease-resistant and yield-increasing of livestock and poultry |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103184174B (en) | 2014-11-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103184174B (en) | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium | |
| CN103820363B (en) | A kind of preparation and application of faecium bacterium powder | |
| CN101690545B (en) | Method for producing complex micro-ecological preparation with microbial agents and enzyme | |
| CN102599351B (en) | Bio-enzyme and microorganism containing compound feed additive and preparation process thereof | |
| CN103173371B (en) | Production of saccharomyces cerevisiae and lactobacillus acidophilus composite microbe preparation used for feed | |
| CN103468594B (en) | Candidautilis strain and application thereof | |
| CN106260504B (en) | Method for producing microbial fermentation wet feed by using beer yeast paste | |
| CN102286376B (en) | Microbial inoculum for high-efficiency fermenting bed and preparation method thereof | |
| CN105432935A (en) | Production method for functional amino-acid humic-acid microecological preparation for aquatic animal and poultry | |
| CN103798540A (en) | Multi-strain microorganism pig feed additive and production method thereof | |
| CN102924134A (en) | Alga microorganism fertilizer agent and preparation method thereof | |
| CN101974463B (en) | Lactobacillus reuteri and composite viable bacteria preparation thereof | |
| CN103160455A (en) | Preparation method of spore preparation of bacillus coagulans | |
| CN101914445B (en) | Indigenous probiotic microorganism solid fungicide and preparation method and application thereof | |
| CN107801837B (en) | Biological fermentation process for forage-based utilization of chicken manure | |
| CN101269884A (en) | Microorganism renovation agent of water environment and preparation method thereof | |
| CN103305434A (en) | Microecological preparation with dual functions of probiotics and organic selenium and preparation method of microecological preparation | |
| CN102719380B (en) | Aquatic compound microecologics and preparation method thereof | |
| CN105002116A (en) | Microorganism straw feed microbial agent and preparation method thereof | |
| CN102409007A (en) | Bacillus microecological preparation and liquid-solid fermentation combining preparation process thereof | |
| CN104293711A (en) | Sheep-and-cow dung fermented compound bacteria, preparation method thereof and method for preparing organic fertilizer by utilizing fermentation of compound bacteria | |
| CN108148791A (en) | A kind of probiotics preparation for improving water quality in aquaculture and preparation method thereof | |
| CN107279462A (en) | The method of chrysanthemum meal fermenting and producing aquatic products livestock and poultry active peptide powder | |
| CN104206645A (en) | Method for producing small peptide feed additive by solid fermentation of soybean meal with aspergillus oryzae | |
| CN103289935A (en) | Compound strain microecological preparation and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |