CN103952336B - Bacillus licheniformis, bacillus subtilis and lactobacillus casei preparation and preparation - Google Patents
Bacillus licheniformis, bacillus subtilis and lactobacillus casei preparation and preparation Download PDFInfo
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Abstract
The invention discloses preparation of a kind of Bacillus licheniformis, bacillus subtilis and lactobacillus casei composite probiotics preparations and preparation method thereof, belong to feed microbe technical field.Described probiotics, with bean cake, wheat bran, Semen Maydis powder for substrate, utilizes Bacillus licheniformis, bacillus subtilis and lactobacillus casei to carry out solid fermentation and forms.The preparation method production cost of the present invention is low, it is possible to increase economic efficiency and simple to operate, can be widely applied to the probiotic solid fermentation technology in Tiny ecosystem field；Adopt mixed culture solid state fermentation, utilize the mutualism synergism between bacterial strain, the viable count that can improve productivity and cell concentration gained fermented product is higher, is conducive to the protein in bean cake and Semen Maydis powder to be broken down into little peptide and free amino acid, is beneficial to digesting and assimilating of the intestines and stomach.When invention product adds use in feedstuff, the utilization ratio 13-25% of feedstuff when this series products is for Cow-feeding, can be effectively improved.
Description
Technical field
The invention belongs to feed microbe technical field, relate to a kind of with bean cake, wheat bran and Semen Maydis powder for substrate, the solid-state fermentation technology being strain with probiotic bacteria Bacillus licheniformis BL-09 (BacilluslicheniformisBL-09), bacillus subtilis HM-66 (BacillussubtilisHM-66) and lactobacillus casei HM-09 (LactobacilluscaseiHM-09), this technology can be widely applied to the aspects such as animal microecological.
Background technology
At present, probiotic composition many employings probiotic bacteria directly with liquid Lac Bovis seu Bubali and soya-bean milk fermentation, obtain functional clabber or sour bean milk, all the other then adopt liquid fermentation, and directly collection probiotic bacteria thalline, is fabricated to probiotic bacteria powder, tablet or capsule.Adopting liquid fermentation can produce substantial amounts of waste water, environmental pollution is relatively big, and post processing is relatively difficult, machinery equipment complex operation.Therefore the application adopts Bacillus licheniformis BL-09, bacillus subtilis HM-66 and lactobacillus casei HM-09 mixed solid fermentation bean cake, wheat bran and Semen Maydis powder, adopt solid fermentation, its production cost is low, the fund of investment is less, convenient in downstream, polluting little, device structure is simply, easily operate.Adopt Bacillus licheniformis BL-09, bacillus subtilis HM-66 and lactobacillus casei HM-09 to carry out solid fermentation, the palatability improving animal feed can be given birth to.Its protein is broken down into little peptide and free amino acid, is conducive to animal intestine peptic digestion to absorb.
Chinese invention patent 200810175856.7 discloses the high density fermentation technology that a strain has the lactobacillus casei (LACTOBACILLUSCASEIZHANG) of beneficial function, control including proliferated culture medium and optimization of fermentation condition, and the technology of preparing of the high vigor lyophilizing mycopowder of this bacterial strain.The lyophilizing mycopowder of the lactobacillus casei of more than 1011CFU/ML viable count can be obtained, it is possible to directly apply to preparation and the application of this bacterial strain probiotics leaven and probiotics preparation by the present invention.
Name is called a kind of lactobacillus casei and the application Chinese invention patent in solid fermentation thereof, and application number is 200710118485.4, this disclosure of the invention a kind of lactobacillus casei and the application in solid fermentation thereof.Described lactobacillus casei (LACTOBACILLUSCASEIZHANG) is the probiotic bacteria separating the acidproof and resistance to bile acid obtained from koumiss；This bacteria strain has been preserved in China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, preserving number: CGMCCNO.1697.The lactobacillus casei (LACTOBACILLUSCASEIZHANG) of the present invention can realize the application in solid fermentation, especially as leaven application in milk product, health food, microbiological feed and animal health goods；The method that the lactobacillus casei thalline survival rate of the present invention improves, is a kind of method optimizing solid medium, can obtain higher cell density and thalline survival rate；The lactobacillus casei solid fermentation viable count of the present invention is more than 20 times of liquid MRS fermentation viable count.
Name is called the lactobacillus casei for feed additive probiotic solid fermentation, animal bifidobacteria, the patent of invention of Lactobacillus plantarum and bacillus subtilis, application number is 01310321977.9, this disclosure of the invention is for the lactobacillus casei of feed additive probiotic solid fermentation, animal bifidobacteria, Lactobacillus plantarum and bacillus subtilis, it is characterized in that: described bacillus subtilis HM-66, lactobacillus casei F-08, Lactobacillus plantarum HM-20 and animal bifidobacteria V9 is probiotic bacteria, it is preserved in China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, preserving number is CGMCCNo.6733 respectively, CGMCCNo.6735, CGMCCNo.6744 and CGMCCNo.5470, bacillus subtilis HM-66, the preservation date of lactobacillus casei F-08 and Lactobacillus plantarum HM-20 is on October 29th, 2012, the preservation date of animal bifidobacteria V9 is on November 18th, 2011.The invention also discloses its application process.
Chinese invention patent for the animal bifidobacteria of feed additive probiotic solid fermentation and bacillus subtilis, application number is 201310321979.8, and invention relates to a kind of fermented type bean cake and the probiotic solid fermentation that can be used for food and feed additive field containing high viable count and high protein degradation rate produced and have special acid fragrance.Bacillus subtilis HM-66 and animal bifidobacteria V9 for solid fermentation are probiotic bacteria, are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNo.6733 and CGMCCNo.5470 respectively.
Chinese invention patent in examining for the Lactobacillus plantarum of probiotic solid fermentation of feed additive field and bacillus subtilis, application number is 201310322177.9, disclose a kind of pollution little, easily operation, be conducive to the Lactobacillus plantarum for feed additive probiotic solid fermentation and bacillus subtilis that gastrointestinal disturbances absorbs, it is characterized in that: described bacillus subtilis HM-66 and Lactobacillus plantarum HM-20 is probiotic bacteria, it is preserved in China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, preserving number is CGMCCNo.6733 and CGMCCNo.6744 respectively.
Chinese invention patent in examining for the lactobacillus casei of probiotic solid fermentation of feed additive field and bacillus subtilis, application number is 201310322953.5.Disclose and a kind of pollute operation little, easy, the bacillus subtilis for feed additive probiotic solid fermentation being conducive to gastrointestinal disturbances to absorb and lactobacillus casei, it is characterized in that: described bacillus subtilis HM-66) and lactobacillus casei F-08 be probiotic bacteria, being preserved in China Committee for Culture Collection of Microorganisms's General Microbiological Culture preservation center, preserving number is CGMCCNo.6733 and CGMCCNo.6735 respectively.
Name is called a kind of effective prevention and improves the compound lactobacillus microbial ecological agent Chinese patent of Mammitis of cattle, application number is 201310094615.0, a kind of compound micro-ecological preparation preventing and improve Mammitis of cattle of this patent disclosure, in compound micro-ecological preparation, lactobacillus casei HM-09 (L.caseiHM-09) mass fraction is 10%, Lactobacillus plantarum HM-10 (L.plantarumHM-10) mass fraction is 10%, all the other 80% are zeolite powder, L.caseiHM-09 number of viable >=2.5 × 108cfu/g in compound micro-ecological preparation simultaneously, L.plantarumHM-10 number of viable >=2.5 × 108cfu/g；Described lactobacillus casei and Lactobacillus plantarum have been great Tun road, Beijing on October 29th, 2012 in address, the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, lactobacillus casei HM-09 (L.caseiHM-09) preserving number: CGMCCNo.6736；Lactobacillus plantarum HM-10 (L.plantarumHM-10) preserving number: CGMCCNo.6741.
Summary of the invention
Present invention aim at adopting Bacillus licheniformis BL-09, bacillus subtilis HM-66 and lactobacillus casei HM-09 to carry out solid fermentation bean cake, wheat bran and Semen Maydis powder, a kind of probiotic solid fermentation technology that can be widely applied to Tiny ecosystem field with bean cake, wheat bran and Semen Maydis powder for substrate is provided, the viable count of the method fermented product is high, and the degradation rate of bean cake protein is high.This preparation method production cost is low, it is possible to increase economic efficiency and simple to operate.
The present invention is achieved through the following technical solutions.
A kind of probiotic solid fermentation technology that can be widely applied to Tiny ecosystem field with bean cake, wheat bran and Semen Maydis powder for substrate, including cultivation and the solid state fermentation culture thereof of Bacillus licheniformis BL-09, bacillus subtilis HM-66 and lactobacillus casei HM-09, obtain the solid fermentation bean cake of probiotic bacteria, wheat bran and Semen Maydis powder product.
Described Bacillus licheniformis BL-09, bacillus subtilis HM-66 and lactobacillus casei HM-09 seed culture medium be: peptone 1-2%, glucose 2-4%, adds water to 100%, pH7.0-7.2.
Described Bacillus licheniformis BL-09, bacillus subtilis HM-66 seed culture condition, be positioned on shaking table, and rotating speed is 150-180r/min, and temperature is 30-37 DEG C, cultivates 20-24h；Lactobacillus casei HM-09 seed culture condition, 30-37 DEG C of quiescent culture 20-24h.
Described solid state fermentation culture method is: first inoculate Bacillus licheniformis BL-09 and bacillus subtilis HM-66 seed liquor in solid-state fermentation culture medium, inoculum concentration is respectively 2-8% (v/w), control fermentation temperature 30-38 DEG C, humidity 40-65%, Hybrid NC machine tool 12-18h；Adding lactobacillus casei HM-09 seed liquor again, inoculum concentration is 1-8% (v/w), and other conditions are constant, then the 12-36h that ferments.Dry acquisition after fermentation ends.
Solid-state fermentation culture medium consists of bean cake, wheat bran and Semen Maydis powder, and composition and the mass ratio ratio of described bean cake, wheat bran and Semen Maydis powder be: bean cake, wheat bran, Semen Maydis powder=10:8-10:0.5-1.Water content controls at 40-75%.
Bacillus licheniformis BL-09, bacillus subtilis HM-66 and lactobacillus casei HM-09 mixed fungus fermentation, after fermentation ends, free ammonical nitrogen content reaches 1109.1umol/g, relatively improving about 11 times before fermentation, Bacillus licheniformis BL-09 and bacillus subtilis HM-66 spore rate are more individually fermented and are respectively increased 2.1 times and 1.5 times.
Bacillus subtilis HM-66 and lactobacillus casei HM-09 was in preservation on October 29 in 2012, and preserving number is CGMCCNo.6733 and CGMCCNo.6736 respectively.Bacterium preservation address is: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Bacillus licheniformis BL-09 specific name respectively (Bacilluslicheniformis) BL-09, Bacillus licheniformis BL-09 are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 31st in December in 2011, bacterium preservation address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode 100101；Preserving number is CGMCCNo.5686.
Bacillus licheniformis BL-09 is isolatable from bacillus cereus in the health cattle intestinal samples of area, Hohhot City ,Inner Mongolia Autonomous Region, belongs to amphimicrobian, Gram-positive bacillus cereus；Thalline is bar shape, is good at shaft-like once in a while, raw in spore, ovalize；Optimum growth temperature is 37 ± 1 DEG C, and the most suitable growth pH is 7.0-7.4, and solid fermentation dries product normal temperature storage 6 months, and spore loss rate is lower than 5%；Nutrient agar panel cultivates about 12h, bacterium colony is irregular, edge is rough, bacterium colony central authorities have significantly protruding peak, it is cultured to 24-36h, bacterium colony fold is obvious and in fold, material is liquid, and inoculating loop picking has wire drawing situation, and microscopy spore comes off substantially, it is defined as Bacillus licheniformis, called after Bacillus licheniformis BL-09 through uncle's Jie Shi antibacterial handbook (the 9th edition) physiological and biochemical test and antibacterial, colonial morphology.Experiment in vitro confirms, this bacterial strain has the biochemical characteristic Bacillus licheniformis BL-09 such as good high temperature resistant, acidproof, bile tolerance and also has the several physiological active substances such as the multiple enzyme such as high proteinase yield, amylase, lipase and aminoacid, vitamin, antibiotic substance, solid medium viscosity after fermentation is very big, wire drawing is serious, visible a small amount of liquid substance bottom culture vessel, it was demonstrated that the moisture-retaining capacity in its solid fermentation is relatively strong.
Acid resistance test:
Bacillus licheniformis BL-09 is in meat soup seed liquor, 37 DEG C, 180r/min cultivates 24h, in 4 DEG C, the centrifugal 10min collection thalline of 4000r/min, with 0.85% physiological saline solution rinsing once after, be suspended in pH respectively 2.0,3.0 and 4.0 buffer solution in, 37 DEG C cultivate 1～3h, detection viable count change, result display Bacillus licheniformis BL-09 has good acid resistance.
Heat resistant test:
Bacillus licheniformis BL-09 is in meat soup seed liquor, 37 DEG C, 180r/min cultivates 24h, in 4 DEG C, the centrifugal 10min collection thalline of 4000r/min, after 0.85% physiological saline solution rinsing once, it is thus achieved that thalline with after the dilution of appropriate physiological saline solution in 80 DEG C of heating in water bath process 10min to obtain spore, then at 80 DEG C, 90 DEG C and 100 DEG C, process 5～30min respectively, detection viable count change, result display Bacillus licheniformis BL-09 has good thermostability.
Bile tolerance is tested:
Bacillus licheniformis BL-09 is in meat soup seed liquor, 37 DEG C, 180r/min cultivates 24h, in 4 DEG C, the centrifugal 10min collection thalline of 4000r/min, with 0.85% physiological saline solution rinsing once after, be suspended in containing gallbladder salinity respectively 0.03%, 0.10%, 0.30% normal saline in, 37 DEG C cultivate 1～6h, detection viable count change, result display Bacillus licheniformis BL-09 has good resistance to light salt.
Beneficial effect:
The present invention adopt Bacillus licheniformis BL-09, bacillus subtilis HM-66 and lactobacillus casei HM-09 mixing carry out solid fermentation bean cake stage by stage.The present invention adopts solid fermentation production cost low, and the fund of investment is less, convenient in downstream, pollutes little, and device structure is simply, easily operate；Lactobacillus casei HM-09 is adopted to carry out mixed fermentation, the tart flavour small-molecule substance such as lactic acid and acetic acid can be produced, these small-molecule substances give improvement and the variation of the organoleptic features such as product distinctive acid fragrance, color and luster and mouthfeel, and fermented product improves animal palatability；In mixed culture solid state fermentation, the mutualism synergism between bacterial strain, can improve productivity and cell concentration；Many bacterial strains produce enzyme, produce acid and produce nutrient substance ability, capacity of decomposition etc. to substrate to be significantly better than single bacterium fermentation；Mixed culture solid state fermentation also has the product that acquisition pure-blood ferment cannot obtain, and fast growth, to substrate utilization ratio height, can multi-stage conversion, the micropopulation after fermentation is stable, and saving of labor is energy-conservation, Simplified flowsheet and Zymolysis Equipment and the plurality of advantages such as easy to control, and not easily microbiological contamination；Adopt Bacillus licheniformis BL-09, bacillus subtilis HM-66 fermented bean cake, wheat bran and Semen Maydis powder, it is possible to make the protein in bean cake and Semen Maydis powder be broken down into little peptide and free amino acid, be beneficial to digesting and assimilating of the intestines and stomach.
In the mechanism of action; the spore of bacillus subtilis and Bacillus licheniformis can be sprouted rapidly after entering host intestine, be bred, and consumes enteral oxygen, it is suppressed that harmful growth needing bacterium; and protect, promote the growth of probiotic bacteria, to safeguard that intestinal microecology balances.Lactobacillus casei can surely be grown in host intestine and lactose can be utilized to produce lactic acid, the Asian lactose intolerance symptom of effective alleviation.The lactic acid energy regulating intestinal canal pH produced, kills not acid resistance pathogen and the hydrogen peroxide produced, bacteriocin etc., other cause of diseases or putrefactive microorganisms has significant inhibitory action, and on probiotic bacteria without impact, thus maintaining intestinal microbial population balance；Can the secretion of induction of immunity globulin A, improve the immunologic function of host, the symptom of colitis can be alleviated by the adjustment to interleukin；Oligopeptide and little peptide that lactobacillus casei fermented milk produces are inhibited to angiotensin, and then play hypotensive activity；Often take lactobacillus casei active bacteria formulation, the cholesterol in blood can be reduced.Bacillus subtilis, Bacillus licheniformis and lactobacillus casei composite probiotics preparations that the present invention obtains, with function above, have good application development prospect.When making that product of the present invention adds use in feedstuff, the utilization ratio 13-25% of feedstuff when this series products is for Cow-feeding, can be effectively improved.The product of the present invention addition in feedstuff is 20-50 gram/kg.
After product of the present invention uses, the conception rate of milch cow improves more than 12%, is computed improving the year milk yield more than 210 kilograms of milch cow.
Detailed description of the invention
Below in conjunction with example, the present invention is further described, and following example is illustrative, is not determinate, it is impossible to limits protection scope of the present invention with following example.
1. prepared by the seed liquor of thalline
Bacillus licheniformis BL-09, bacillus subtilis HM-66 and lactobacillus casei HM-09 receive equipped with in the culture medium of liquid seeds from the glycerol pipe of cold storage, Bacillus licheniformis BL-09, bacillus subtilis HM-66 seed culture condition, it is positioned on shaking table, rotating speed is 150r/min, temperature is 37 DEG C, cultivates 24h；Lactobacillus casei HM-09 seed culture condition, 37 DEG C of quiescent culture 24h.
Wherein liquid seed culture medium (g/L) is: peptone 1%, and glucose 2% adds water to 100%, pH7.0-7.2.
2. solid fermentation
First inoculating Bacillus licheniformis BL-09 and bacillus subtilis HM-66 seed liquor in solid-state fermentation culture medium, inoculum concentration is respectively 5% (v/w), controls fermentation temperature 35 DEG C, humidity 55%, Hybrid NC machine tool 12h；Adding lactobacillus casei HM-09 seed liquor again, inoculum concentration is 4% (v/w), and other conditions are constant, then the 24h that ferments.Fermentation ends obtains dry culture through fluidized bed drying method.
Solid-state fermentation culture medium is: bean cake, wheat bran, Semen Maydis powder=10:8:0.5.Moisture 40%, 115 DEG C of autoclaving 25min.
3. measure viable count
It is generally adopted colony counting method, operating procedure is: weigh the bean pulp fermentation goods 10g prepared, it is placed in the physiological saline solution of 90ml, it is placed on shaking table and shakes 30min, take in the physiological saline solution that the bacterium solution of 1ml adds another 9ml with liquid-transfering gun after shaking up, it is diluted, according to national standard method, takes turns doing ten times of gradient dilutions.After having diluted, take suitable dilution factor 1ml with liquid-transfering gun respectively and throw flat board into, pour into culture medium mixing, take turns doing three parallel, be placed in incubator at 37 DEG C of quiescent culture 48h.
The mensuration of 4.pH value
Weigh the product after 10g fermentation, put in triangular flask, add the distilled water of 90ml.Use magnetic stirrer 30min, after standing 10min, determine its pH value with Accurate pH measurement.
5. the assay method of amino-acid nitrogen content
Take the product after 10g fermentation, crush by homogenizer, mix with the distilled water of 90ml afterwards, place 1 day at 4 DEG C of refrigerators.Sample is centrifuged 7000r/min, centrifugal 15min.Supernatant 0.8ml is placed in test tube, then adds 3.2ml distilled water, 2.0ml developer, mixing, puts into heating 15min in boiling water bath, makes blank simultaneously.Then cold water cooling, adds the mixing of 5.0ml40% alcoholic solution, uses 1cm cuvette after placing 15min, and return to zero with blank tube measures A value in 570nm place.
The following is the determination of some parameters of production method to the probiotics fermention product being substrate with bean cake and the optimization of some condition of culture.
Embodiment 1:
Described solid state fermentation culture method is: first inoculate Bacillus licheniformis BL-09 and bacillus subtilis HM-66 seed liquor in solid-state fermentation culture medium, and inoculum concentration is respectively 3% (v/w), controls fermentation temperature 35 DEG C, humidity 60%, Hybrid NC machine tool 12h；Adding lactobacillus casei HM-09 seed liquor again, inoculum concentration is 5% (v/w), and other conditions are constant, then the 12h that ferments, and fermentation total time is 24h altogether.Fermentation ends obtains dry culture through fluidized bed drying method.
Solid-state fermentation culture medium consists of bean cake, wheat bran and Semen Maydis powder, and composition and the mass ratio ratio of described bean cake, wheat bran and Semen Maydis powder be: bean cake, wheat bran, Semen Maydis powder=10:10:0.5, water content controls 60%.
Recording lactobacillus casei HM-66 viable count is 4.05 × 109Cfu/g, HM-09 bacillus subtilis HM-66 viable count is 1.05 × 1010Cfu/g, Bacillus licheniformis BL-09 viable count is 1.29 × 1010Cfu/g, after fermentation ends, free ammonical nitrogen content reaches 1038.7umol/g.
Embodiment 2:
Described solid state fermentation culture method is: first inoculate Bacillus licheniformis BL-09 and bacillus subtilis HM-66 seed liquor in solid-state fermentation culture medium, and inoculum concentration is respectively 3% (v/w), controls fermentation temperature 37 DEG C, humidity 40%, Hybrid NC machine tool 15h；Adding lactobacillus casei HM-09 seed liquor again, inoculum concentration is 5% (v/w), and other conditions are constant, then the 16h that ferments, and fermentation ends obtains dry culture through fluidized bed drying method.
Solid-state fermentation culture medium consists of bean cake, wheat bran and Semen Maydis powder, and composition and the mass ratio ratio of described bean cake, wheat bran and Semen Maydis powder be: bean cake, wheat bran, Semen Maydis powder=10:8:1, water content controls 70%.
Recording lactobacillus casei HM-09 viable count is 4.09 × 109Cfu/g, bacillus subtilis HM-66 viable count is 0.95 × 1010Cfu/g, Bacillus licheniformis BL-09 viable count is 1.18 × 1010Cfu/g, after fermentation ends, free ammonical nitrogen content reaches 1019.9umol/g.
Embodiment 3:
Described solid state fermentation culture method is: first inoculate Bacillus licheniformis BL-09 and bacillus subtilis HM-66 seed liquor in solid-state fermentation culture medium, and inoculum concentration is respectively 5% (v/w), controls fermentation temperature 30 DEG C, humidity 65%, Hybrid NC machine tool 18h；Adding lactobacillus casei HM-09 seed liquor again, inoculum concentration is 5% (v/w), and other conditions are constant, then the 18h that ferments, and fermentation ends obtains dry culture through fluidized bed drying method.
Solid-state fermentation culture medium consists of bean cake, wheat bran and Semen Maydis powder, and composition and the mass ratio ratio of described bean cake, wheat bran and Semen Maydis powder be: bean cake, wheat bran, Semen Maydis powder=10:9:0.8, water content controls 70%.
Recording lactobacillus casei HM-09 viable count is 4.10 × 109Cfu/g, bacillus subtilis HM-66 viable count is 0.81 ×1010cfu/g, Bacillus licheniformis BL-09 viable count is 1.05 ×1010cfu/g, after fermentation ends, free ammonical nitrogen content reaches 1074.5umol/g.
Embodiment 4
As Bacillus licheniformis BL-09, bacillus subtilis HM-66 and lactobacillus casei HM-09 inoculum concentration respectively 3.0% (v/w), 3.0% (v/w) and 1.0% (v/w), and first with after Bacillus licheniformis BL-09 and bacillus subtilis HM-66 mixed fungus fermentation 12h, again with lactobacillus casei HM-09 mixed fungus fermentation 36h, cultivation temperature 37 ± 1 DEG C, amount to incubation time 48h, its viable count is higher, and recording lactobacillus casei HM-09 viable count is 4.55 × 109Cfu/g, bacillus subtilis HM-66 viable count is 1.35 × 1010Cfu/g, Bacillus licheniformis BL-09 viable count is 1.55 × 1010Cfu/g, and the content of now free ammonical nitrogen is the highest, reaches 1109.1umol/g.
Claims (4)
1. a composite probiotics preparations, prepared by following method: first solid-state fermentation culture medium is inoculated Bacillus licheniformis BL-09, preserving number is CGMCCNo.5686 and bacillus subtilis HM-66 seed liquor, inoculum concentration is respectively 2-8% (v/w), control fermentation temperature 30-38 DEG C, humidity 40-65%, Hybrid NC machine tool 12-18h；Adding lactobacillus casei HM-09 seed liquor again, inoculum concentration is 1-8% (v/w), and other conditions are constant, then the 12-36h that ferments, and fermentation ends dries.
2. composite probiotics preparations according to claim 1: it is characterized in that, solid-state fermentation culture medium consists of bean cake, wheat bran and Semen Maydis powder, and the mass ratio of described bean cake, wheat bran and Semen Maydis powder is: bean cake, wheat bran, Semen Maydis powder=10:8-10:0.5-1.
3. the preparation method of composite probiotics preparations according to claim 1, comprise the steps: first solid-state fermentation culture medium is inoculated Bacillus licheniformis BL-09, preserving number is CGMCCNo.5686 and bacillus subtilis HM-66 seed liquor, inoculum concentration is respectively 2-8% (v/w), control fermentation temperature 30-38 DEG C, humidity 40-65%, Hybrid NC machine tool 12-18h；Adding lactobacillus casei HM-09 seed liquor again, inoculum concentration is 1-8% (v/w), and other conditions are constant, then the 12-36h that ferments, and fermentation ends dries；Solid-state fermentation culture medium consists of bean cake, wheat bran and Semen Maydis powder, and the mass ratio of described bean cake, wheat bran and Semen Maydis powder is: bean cake, wheat bran, Semen Maydis powder=10:8-10:0.5-1.
4. the composite probiotics preparations according to claim 1 and 2 application in livestock and poultry cultivation.
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| CN105602865B (en) * | 2015-12-23 | 2019-07-09 | 河南广安生物科技股份有限公司 | One bacillus subtilis, fermentation process, pulvis and application |
| CA3025661A1 (en) | 2016-05-29 | 2017-12-07 | The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) | Method of generating bacterial compositions |
| CN105994990A (en) * | 2016-05-31 | 2016-10-12 | 灵宝市帅华牧业有限责任公司 | Preparation method of folium cortex eucommiae biologically fermented compound bacterium feed additive |
| CN107296164A (en) * | 2017-07-19 | 2017-10-27 | 东北农业大学 | A kind of preparation method for the Traditional Chinese medicine probiotic fermentating metabolism product for preventing and treating piglet bacillary diarrhoea |
| CN113424900A (en) * | 2021-07-12 | 2021-09-24 | 广西壮族自治区兽医研究所 | Preparation method, product and application of probiotic feed additive |
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| CN102229900A (en) * | 2011-05-20 | 2011-11-02 | 周剑平 | Compound probiotics preparation for biological organic fertilizers and preparation method thereof |
| CN103202391A (en) * | 2013-03-22 | 2013-07-17 | 内蒙古和美科盛生物技术有限公司 | Compound lactobacillus probiotics for effectively preventing and improving bovine mastitis |
| CN103211085A (en) * | 2013-04-23 | 2013-07-24 | 保龄宝生物股份有限公司 | Efficient feed compound probiotics preparation and preparation method thereof |
| CN103421711A (en) * | 2013-07-29 | 2013-12-04 | 内蒙古和美科盛生物技术有限公司 | Lactobacillus casei, bifidobacterium animalis, lactobacrillus plantarum and bacillus subtilis for solid fermentation of probiotics in field of feed additives |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229900A (en) * | 2011-05-20 | 2011-11-02 | 周剑平 | Compound probiotics preparation for biological organic fertilizers and preparation method thereof |
| CN103202391A (en) * | 2013-03-22 | 2013-07-17 | 内蒙古和美科盛生物技术有限公司 | Compound lactobacillus probiotics for effectively preventing and improving bovine mastitis |
| CN103211085A (en) * | 2013-04-23 | 2013-07-24 | 保龄宝生物股份有限公司 | Efficient feed compound probiotics preparation and preparation method thereof |
| CN103421711A (en) * | 2013-07-29 | 2013-12-04 | 内蒙古和美科盛生物技术有限公司 | Lactobacillus casei, bifidobacterium animalis, lactobacrillus plantarum and bacillus subtilis for solid fermentation of probiotics in field of feed additives |
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