CN103911323A - Bacillus licheniformis, bacillus subtilis and lactobacillus plantarum preparation and preparation - Google Patents
Bacillus licheniformis, bacillus subtilis and lactobacillus plantarum preparation and preparation Download PDFInfo
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Abstract
The invention belongs to the micro biotechnical field of feed and relates to a probiotics preparation which is prepared by solid fermentation of probiotics bacillus licheniformis BL-09, bacillus subtilis HM-66 and lactobacillus plantarum HM-10 which are mixed in stages by taking bean pulp, bran and corn flour as a matrix. According to the invention, the yield and cell concentration can be improved by adopting mixed bacteria solid state fermentation. The ability of multiple strains to yield enzymes, acids and nutritional substances and the capacity of decomposition to the matrix and the like are remarkably superior to single bacterial fermentation. The product which cannot be obtained by pure fermentation can be obtained, the preparation has the advantages of fast growing speed, high primer utilization ratio, conversion in multiple stages, stable fermented microbial population, labor and energy saving, simplified process and fermentation equipment, easiness in control and the like, and is not easy to contaminate. By fermenting soybean meal, bran and corn flour by the three bacterial strains, proteins in the soybean meal and corn flour can be decomposed to small peptides and free amino acids to facilitate digestive absorption by intestines and stomach.
Description
Technical field
The invention belongs to feed microbe technical field, relate to a kind of taking dregs of beans, wheat bran and Semen Maydis powder as matrix, with probiotic bacterium Bacillus licheniformis BL-09(Bacillus licheniformis BL-09), subtilis HM-66(Bacillus subtilis HM-66) and plant lactobacillus HM-10(Lactobacillus plantarum HM-10) probiotics preparation prepared of solid state fermentation and preparation method thereof, can be widely used in the aspects such as animal microecological.
Background technology
At present, probiotic composition adopts liquid state fermentation more, directly collects probiotic bacterium thalline, is made into probiotic bacterium pulvis, tablet or capsule.Adopt liquid state fermentation meeting to produce a large amount of waste water, environmental pollution is larger, and aftertreatment is more difficult, machinery equipment complex operation.How to obtain the bacterial strain that is applicable to solid state fermentation, preparing the probiotics preparation that number of viable is high and vigor is good will become the direction of industry development.The application adopts Bacillus licheniformis BL-09, subtilis HM-66 and plant lactobacillus HM-10 mixed solid fermentation dregs of beans, wheat bran and Semen Maydis powder, adopts solid state fermentation, and its production cost is low, the fund of investment is less, convenient in downstream processing, pollute littlely, device structure is simple, easy to operate.Adopt Bacillus licheniformis BL-09, subtilis HM-66 and plant lactobacillus HM-10 to carry out solid state fermentation, can give birth to the palatability of improving animal-feed.Its protein is broken down into little peptide and total free aminoacids, is conducive to animal intestine peptic digestion and absorbs.
Publication number is that the Chinese invention patent " for plant lactobacillus and the subtilis of the probiotic bacterium solid fermentation of feed additive field " of 103421713A discloses a kind of subtilis HM-66 and the plant lactobacillus HM-20 solid fermentation probiotic bacterium for the preparation of feed additive field, and discloses its application method.When subtilis HM-66 and plant lactobacillus HM-20 mixed fungus fermentation, the suitableeest inoculum size of subtilis is 1%, the suitableeest inoculum size of plant lactobacillus HM-20 is 4%, carry out mixed solid fermentation dregs of beans, can give product distinctive sour fragrance, improve the organoleptic feature such as color and luster and mouthfeel of product, can also make the protein in dregs of beans be broken down into little peptide and total free aminoacids, be beneficial to digesting and assimilating of stomach.Described culture presevation is in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, and preserving number is respectively CGMCC No.6733 and CGMCC No.6744.Preservation date is: on October 29th, 2012.
Publication number is that the Chinese invention patent " for lactobacterium casei, animal bifidobacteria, plant lactobacillus and the subtilis of fodder additives probiotic bacterium solid fermentation " of 103421711A discloses subtilis HM-66, lactobacterium casei F-08, plant lactobacillus HM-20 and animal bifidobacteria V9, for fodder additives probiotic bacterium solid fermentation, and its application method is disclosed.Above-mentioned culture presevation is in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, and preserving number is respectively CGMCCNo.6733, CGMCC No.6735, CGMCC No.6744 and CGMCCNo.5470.
Publication number is that the Chinese invention patent " a kind of effective prevention and improve the compound lactobacillus probiotics of garget " of 103202391A discloses a kind of compound micro-ecological preparation, wherein lactobacterium casei HM-09 mass fraction is 10%, plant lactobacillus HM-10 mass fraction is 10%, all the other 80% are zeolite powder, simultaneously HM-09 number of viable>=2.5 × 10 in compound micro-ecological preparation
8cfu/g, HM-10 number of viable>=2.5 × 10
8cfu/g; Described lactobacterium casei and plant lactobacillus on October 29th, 2012 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, lactobacterium casei HM-09 preserving number: CGMCC No.6736; Plant lactobacillus HM-10 preserving number: CGMCC No.6741.This invention has good prevention and improvement effect to mastitis ox, mastitis sickness rate can be reduced to 84.7%, and sickness rate obviously declines, and improves milk cow health level.
Summary of the invention
The object of the invention is to adopt Bacillus licheniformis BL-09, subtilis HM-66 and plant lactobacillus HM-10 to carry out solid state fermentation dregs of beans, wheat bran and Semen Maydis powder, a kind of probiotics preparation that can be widely used in micro-ecosphere taking dregs of beans, wheat bran and Semen Maydis powder as matrix is provided, and provide the preparation method of this probiotics preparation, the viable count of probiotics preparation prepared by the method is high, nutritive substance degradation rate is high, production cost is low, can increase economic efficiency and simple to operate.
The present invention is achieved through the following technical solutions.
A kind of probiotics preparation that can be widely used in micro-ecosphere taking dregs of beans, wheat bran and Semen Maydis powder as matrix, the preparation method of described probiotics preparation is solid state fermentation, and step comprises cultivation and the solid state fermentation cultivation thereof of Bacillus licheniformis BL-09, subtilis HM-66 and plant lactobacillus HM-10.Described Bacillus licheniformis BL-09, subtilis HM-66 and plant lactobacillus HM-10 seed culture medium mass percent consist of: peptone 1%, glucose 2%, adds water to 100%, pH7.0-7.2.
Solid-state fermentation culture medium consists of dregs of beans, wheat bran and Semen Maydis powder, and the mass ratio of described dregs of beans, wheat bran and Semen Maydis powder is: dregs of beans, wheat bran, Semen Maydis powder=10:9-10:0.5-1, water content is controlled at 60-70%.
Described Bacillus licheniformis BL-09, subtilis HM-66 seed culture condition, be positioned on shaking table, and rotating speed is 150r/min, and temperature is 37 DEG C, cultivates 24h; Plant lactobacillus HM-10 seed culture condition, 37 DEG C leave standstill cultivation 24h.
Described solid state fermentation cultural method is: in solid-state fermentation culture medium, first inoculate Bacillus licheniformis BL-09 and subtilis HM-66 seed liquor, inoculum size is identical, be respectively 3.0-5.0%(v/w), controlled fermentation temperature 30-37 DEG C, humidity 70-80%, mixed bacterium is cultivated 12-24h, add plant lactobacillus HM-10 seed liquor, inoculum size is 1.0-3.0%(v/w again), other conditions are constant, the 12-24h that ferments again, the total time of fermenting is altogether 24-48h.Add after plant lactobacillus HM-10, at the mixed fermentation initial stage, pH is on a declining curve, and pH minimum value is 5.0-6.0, and in the later stage of fermentation, pH is in rising trend, and after fermentation 48h, its pH is 6.5-7.5.
Utilize in the ban after Bacillus licheniformis BL-09 and subtilis HM-66 mixed fungus fermentation 12h, again with plant lactobacillus HM-10 mixed fungus fermentation 36h, ferment altogether after 48h, free ammonical nitrogen content can reach 1136.5umol/g, before fermentation, improve approximately 12 times, Bacillus licheniformis BL-09 and subtilis HM-66 gemma rate are fermented more separately and are improved respectively 2.4 times and 1.8 times.
The preparation method of described probiotics preparation is: after fermentation ends, obtain dry culture through fluidised bed drying.
Product of the present invention makes an addition in feed, during for detoxification, can effectively improve the utilising efficiency 12-30% of feed.During for Cow-feeding, the conception rate of milk cow has improved 12%, can improve as calculated the more than year milk yield 200-300 kilogram of milk cow.The addition of product of the present invention in feed is 20-100 gram/ton.
Bacillus licheniformis BL-09 was in preservation on December 31 in 2011, deposit number is CGMCC No.5686, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.Subtilis HM-66 and plant lactobacillus HM-10 were in preservation on October 29 in 2012, and deposit number is respectively CGMCC No.6733 and CGMCC No.6741.Preservation address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Bacillus licheniformis BL-09 specific name is Bacillus licheniformis BL-09, separates genus bacillus in healthy Roll road, area, Hohhot City ,Inner Mongolia Autonomous Region sample, belongs to amphimicrobian, Gram-positive genus bacillus; It is shaft-like that thalline is rod, be good at once in a while shaft-like, raw in gemma, ovalize; Optimum growth temperature is 37 ± 1 DEG C, and the most suitable growth pH is 7.0-7.4, and solid state fermentation is dried product normal temperature storage 6 months, and gemma rate of loss is lower than 5%; Nutrient agar plate is cultivated 12h left and right, bacterium colony is irregular, edge is rough, bacterium colony central authorities have obvious protruding peak, be cultured to 24-36h, in the obvious and fold of bacterium colony fold, material is liquid, and transfering loop picking has wire drawing situation, and microscopy gemma comes off substantially, process uncle's Jie Shi bacterium handbook (the 9th edition) physiological and biochemical test and bacterium, colonial morphology are defined as Bacillus licheniformis, called after Bacillus licheniformis BL-09.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 amino acid, VITAMIN, antimicrobial substance, solid medium viscosity after fermentation is very large, wire drawing is serious, the visible a small amount of liquid substance in culture vessel bottom, proves that the moisture-retaining capacity in its solid state fermentation is relatively strong.
Acid resistance test:
Bacillus licheniformis BL-09 is in meat soup seed liquor, 37 DEG C, 150r/min cultivates 24h, in 4 DEG C, the centrifugal 10min collection of 4000r/min thalline, with after 0.85% stroke-physiological saline solution rinsing once, be suspended in pH and be respectively in 2.0,3.0 and 4.0 buffered soln, cultivate 1-3h for 37 DEG C, detection viable count changes, and result shows that Bacillus licheniformis BL-09 has good acid resistance.
Oven test:
Bacillus licheniformis BL-09 is in meat soup seed liquor, 37 DEG C, 150r/min cultivates 24h, in 4 DEG C, the centrifugal 10min collection of 4000r/min thalline, with after 0.85% stroke-physiological saline solution rinsing once, after the thalline of acquisition dilutes by appropriate stroke-physiological saline solution, process 10min to obtain gemma in 80 DEG C of heating in water bath, then process respectively 5-30min at 80 DEG C, 90 DEG C and 100 DEG C, detection viable count changes, and result shows that Bacillus licheniformis BL-09 has good thermotolerance.
Bile tolerance 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 of 4000r/min thalline, with after 0.85% stroke-physiological saline solution rinsing once, be suspended in containing gallbladder salinity and be respectively in 0.03%, 0.10%, 0.30% physiological saline, cultivate 1-6h for 37 DEG C, detection viable count changes, and result shows that Bacillus licheniformis BL-09 has good bile tolerance.
Beneficial effect
In the present invention, adopt Bacillus licheniformis BL-09, subtilis HM-66 and plant lactobacillus HM-10 to mix and carry out stage by stage solid state fermentation dregs of beans.The present invention adopts solid state fermentation production cost low, and the fund of investment is less, convenient in downstream processing, pollutes littlely, and device structure is simple, easy to operate; Adopt plant lactobacillus HM-10 to carry out mixed fermentation, can produce the tart flavour such as lactic acid and acetic acid small-molecule substance, these small-molecule substances are given improvement and the variation of the organoleptic features such as the distinctive sour fragrance of product, color and luster and mouthfeel, and leavened prod improves animal palatability; In mixed culture solid state fermentation, the mutualism synergy between bacterial strain, can improve productive rate and cell concentration; Product enzyme, product acid and product nutritive substance ability, the capacity of decomposition to matrix etc. of many bacterial strains will significantly be better than single bacterium fermentation; Mixed culture solid state fermentation also has the product that acquisition pure-blood ferment cannot obtain, and fast growth is high to substrate utilization ratio, can multistagely transform, the microbial population after fermentation is stable, and saving of labor is energy-conservation, simplify technique and fermentation equipment and the plurality of advantages such as easy to control, and be difficult for microbiological contamination; Adopt Bacillus licheniformis BL-09, subtilis HM-66 fermented bean dregs, wheat bran and Semen Maydis powder, can make the protein in dregs of beans and Semen Maydis powder be broken down into little peptide and total free aminoacids, be beneficial to digesting and assimilating of stomach.
Aspect the mechanism of action; the gemma of subtilis and Bacillus licheniformis can be sprouted rapidly, be bred after host endospore enters host's enteron aisle, consumes oxygen in intestines, suppresses harmful growth that needs bacterium; and protect, promote the growth of probiotic bacterium, to safeguard intestinal microecology balance.Plant lactobacillus can surely be grown in host's enteron aisle and can utilize lactose to produce lactic acid, effectively alleviates Asian lactose intolerance symptom.Produce lactic acid energy regulating intestinal canal pH, kill not acid resistance pathogenic bacteria, and produce hydrogen peroxide, bacteriocin etc., other cause of diseases or spoilage microorganisms are had to significant restraining effect, and on probiotic bacterium without impact, thereby maintain intestinal microflora balance; Secretion that can induction of immunity globulin A, improves host's immunologic function, can be by the adjusting of interleukin being alleviated to the symptom of colitis; Oligopeptides and little peptide that plant lactobacillus fermented-milk produces are inhibited to Angiotensin, and then play hypotensive activity; Often take plant lactobacillus active bacteria formulation, can reduce the cholesterol in blood.Subtilis, Bacillus licheniformis and plant lactobacillus composite probiotics preparations that this patent obtains, with above function, have good application development prospect.
Embodiment
Below in conjunction with example, the present invention is further described, and following example is illustrative, is not determinate, can not limit protection scope of the present invention with following example.
Bacillus licheniformis BL-09, subtilis HM-66 and plant lactobacillus HM-10, specific name is respectively Bacillus licheniformis BL-09, Bacillus subtilis HM-66 and Lactobacillus plantarum HM-10.Bacillus licheniformis BL-09 was in preservation on December 31 in 2011, and preserving number is CGMCC No.5686; Subtilis HM-66 and plant lactobacillus HM-10 were in preservation on October 29 in 2012, and preserving number is respectively CGMCC No.6733 and CGMCC No.6741.3 strain bacterium preservation addresses are: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center.
1. measure viable count
The general colony counting method that adopts, operation steps is: take the bean pulp fermentation goods 10g preparing, be placed in the stroke-physiological saline solution of 90ml, be placed into and on shaking table, shake 30min, shake up and add in the stroke-physiological saline solution of another 9ml with the bacterium liquid that liquid-transfering gun is got 1ml afterwards, dilute, according to national standard method, take turns doing ten times of gradient dilutions.After having diluted, get respectively suitable extent of dilution 1ml with liquid-transfering gun and throw flat board into, pour substratum into and mix, take turns doing three parallel, be placed in incubator 37 DEG C leave standstill cultivate 48h.
The mensuration of 2.pH value
Take the product after 10g fermentation, put into triangular flask, add the distilled water of 90ml.Use magnetic stirrer 30min, after standing 10min, determine its pH value with Accurate pH instrumentation.
3. the measuring method of amino-acid nitrogen content
Get the product after 10g fermentation, use homogenizer fragmentation, mix with the distilled water of 90ml afterwards, place 1 day at 4 DEG C of refrigerators.The centrifugal 7000r/min of sample, centrifugal 15min.Supernatant liquor 0.8ml is placed in test tube, then adds 3.2ml distilled water, 2.0ml developer, mixes, and puts into boiling water bath and heats 15min, makes blank simultaneously.Then cold water is cooling, adds 5.0ml40% ethanolic soln to mix, and after placement 15min, uses 1cm cuvette, returns to zero and measures A value in 570nm place with blank tube.
Embodiment 1:
Probiotics preparation prepared by Bacillus licheniformis, subtilis and plant lactobacillus, its preparation method comprises spawn culture and solid state fermentation cultivation thereof.
Described solid state fermentation cultural method is: in solid-state fermentation culture medium, first inoculate Bacillus licheniformis BL-09 and subtilis HM-66 seed liquor, inoculum size is respectively 3.0% (v/w), 35 DEG C of controlled fermentation temperature, and humidity 75%, mixed bacterium is cultivated 12h; Add plant lactobacillus HM-10 seed liquor, inoculum size is 1.0% (v/w) again, and other conditions are constant, then the 12h that ferments, and the total time of fermenting is altogether 24h.Fermentation ends obtains dry culture through fluidized bed drying method.
Solid-state fermentation culture medium consists of dregs of beans, wheat bran and Semen Maydis powder, and the mass ratio of described dregs of beans, wheat bran and Semen Maydis powder is: dregs of beans, wheat bran, Semen Maydis powder=10:10:0.5, water content is controlled at 60%.
Recording plant lactobacillus HM-10 viable count is 3.00 × 10
9cfu/g, subtilis HM-66 viable count is 1.01 × 10
10cfu/g, Bacillus licheniformis BL-09 viable count is 1.15 × 10
10cfu/g, after fermentation ends, free ammonical nitrogen content reaches 958.1umol/g.
Embodiment 2:
Probiotics preparation prepared by Bacillus licheniformis, subtilis and plant lactobacillus, its preparation method comprises spawn culture and solid state fermentation cultivation thereof.
Described solid state fermentation cultural method is: in solid-state fermentation culture medium, first inoculate Bacillus licheniformis BL-09 and subtilis HM-66 seed liquor, inoculum size is respectively 3.0% (v/w), 37 DEG C of controlled fermentation temperature, and humidity 75%, mixed bacterium is cultivated 12h; Add plant lactobacillus HM-10 seed liquor, inoculum size is 1.0% (v/w) again, and other conditions are constant, then the 24h that ferments, and fermentation ends obtains dry culture through fluidized bed drying method.
Solid-state fermentation culture medium consists of dregs of beans, wheat bran and Semen Maydis powder, and the mass ratio of described dregs of beans, wheat bran and Semen Maydis powder is: dregs of beans, wheat bran, Semen Maydis powder=10:9:1, water content is controlled at 60%.
Recording plant lactobacillus HM-10 viable count is 3.16 × 10
9cfu/g, subtilis HM-66 viable count is 1.16 × 10
10cfu/g, Bacillus licheniformis BL-09 viable count is 1.33 × 10
10cfu/g, after fermentation ends, free ammonical nitrogen content reaches 1073.1umol/g.
Embodiment 3:
Probiotics preparation prepared by Bacillus licheniformis, subtilis and plant lactobacillus, its preparation method comprises spawn culture and solid state fermentation cultivation thereof.
Described solid state fermentation cultural method is: in solid-state fermentation culture medium, first inoculate Bacillus licheniformis BL-09 and subtilis HM-66 seed liquor, inoculum size is respectively 5.0% (v/w), 30 DEG C of controlled fermentation temperature, and humidity 75%, mixed bacterium is cultivated 24h; Add plant lactobacillus HM-10 seed liquor, inoculum size is 3.0% (v/w) again, and other conditions are constant, then the 24h that ferments, and fermentation ends obtains dry culture through fluidized bed drying method.
Solid-state fermentation culture medium consists of dregs of beans, wheat bran and Semen Maydis powder, and the mass ratio of described dregs of beans, wheat bran and Semen Maydis powder is: dregs of beans, wheat bran, Semen Maydis powder=10:9:0.8, water content is controlled at 70%.
Recording plant lactobacillus HM-10 viable count is 2.85 × 10
9cfu/g, subtilis HM-66 viable count is 1.06 × 10
10cfu/g, Bacillus licheniformis BL-09 viable count is 1.10 × 10
10cfu/g, after fermentation ends, free ammonical nitrogen content reaches 1006.0umol/g.
Embodiment 4
As Bacillus licheniformis BL-09, subtilis HM-66 and plant lactobacillus HM-10 inoculum size are respectively 3.0% (v/w), 3.0% (v/w) and 1.0% (v/w), in solid-state fermentation culture medium, first inoculate Bacillus licheniformis BL-09 and subtilis HM-66 seed liquor, first utilize after Bacillus licheniformis BL-09 and subtilis HM-66 mixed fungus fermentation 12h, again with plant lactobacillus HM-10 mixed fungus fermentation 36h, 37 ± 1 DEG C of culture temperature, humidity 75%, amount to incubation time 48h, fermentation ends obtains dry culture through fluidized bed drying method, its viable count is higher, recording plant lactobacillus HM-10 viable count is 3.45 × 10
9cfu/g, subtilis HM-66 viable count is 1.43 × 10
10cfu/g, Bacillus licheniformis BL-09 viable count is 1.65 × 10
10cfu/g, and now the content of free ammonical nitrogen is the highest, reaches 1136.5umol/g.
Solid-state fermentation culture medium consists of dregs of beans, wheat bran and Semen Maydis powder, and the mass ratio of described dregs of beans, wheat bran and Semen Maydis powder is: dregs of beans, wheat bran, Semen Maydis powder=10:10:0.5, water content is controlled at 60%.
Claims (10)
1. a probiotics preparation prepared by Bacillus licheniformis, subtilis and plant lactobacillus, it is characterized in that, adopt Bacillus licheniformis BL-09, subtilis HM-66 and plant lactobacillus HM-10 to mix solid state fermentation stage by stage, after fermentation ends, prepare through fluidised bed drying;
Described Bacillus licheniformis BL-09 deposit number is CGMCCNo.5686;
Described subtilis HM-66 deposit number is CGMCCNo.6733;
Described plant lactobacillus HM-10 deposit number is CGMCCNo.6741.
2. the probiotics preparation that according to claim 1 prepared by Bacillus licheniformis, subtilis and plant lactobacillus, described mixing stage by stage solid state fermentation comprises that the seed culture of Bacillus licheniformis BL-09, subtilis HM-66 and plant lactobacillus HM-10 and solid state fermentation thereof cultivate, it is characterized in that
Described seed culture medium mass percent consists of: peptone 1%, and glucose 2%, adds water to 100%, pH7.0-7.2;
Described solid-state fermentation culture medium mass ratio consists of: dregs of beans: wheat bran: Semen Maydis powder=10:9-10:0.5-1, water content is controlled at 60-70%.
3. the probiotics preparation that according to claim 1 prepared by Bacillus licheniformis, subtilis and plant lactobacillus, described Bacillus licheniformis BL-09, subtilis HM-66 seed culture condition, be positioned on shaking table, and rotating speed is 150r/min, temperature is 37 DEG C, cultivates 24h; Plant lactobacillus HM-10 seed culture condition, 37 DEG C leave standstill cultivation 24h.
4. Bacillus licheniformis according to claim 1, probiotics preparation prepared by subtilis and plant lactobacillus, described solid state fermentation cultural method is: in solid-state fermentation culture medium, first inoculate Bacillus licheniformis BL-09 and subtilis HM-66 seed liquor, inoculum size is identical, be respectively 3.0-5.0%(v/w), controlled fermentation temperature 30-37 DEG C, humidity 70-80%, mixed bacterium is cultivated 12-24h, add again plant lactobacillus HM-10 seed liquor, inoculum size is 1.0-3.0%(v/w), other conditions are constant, 12-24h again ferments, total time of fermenting is altogether 24-48h.
5. the probiotics preparation that according to claim 1 prepared by Bacillus licheniformis, subtilis and plant lactobacillus, prepared by following methods:
1. spawn culture
Bacillus licheniformis BL-09, subtilis HM-66 seed culture condition, be positioned on shaking table, and rotating speed is 150r/min, and temperature is 37 DEG C, cultivates 24h; Plant lactobacillus HM-10 seed culture condition, 37 DEG C leave standstill cultivation 24h;
2. solid state fermentation
In solid-state fermentation culture medium, first inoculate Bacillus licheniformis BL-09 and subtilis HM-66 seed liquor, inoculum size is respectively 3.0% (v/w), 37 ± 1 DEG C of culture temperature, humidity 75%, after fermentation 12h, inoculation 1.0% (v/w) plant lactobacillus HM-10 mixed fungus fermentation 36h;
3. dry
Fermentation ends obtains dry culture through fluidised bed drying.
6. prepare the method for probiotics preparation according to the arbitrary described Bacillus licheniformis of claim 1-5, subtilis and plant lactobacillus, it is characterized in that, adopt Bacillus licheniformis BL-09, subtilis HM-66 and plant lactobacillus HM-10 to mix solid state fermentation stage by stage, after fermentation ends through fluidised bed drying;
Described Bacillus licheniformis BL-09 deposit number is CGMCCNo.5686;
Described subtilis HM-66 deposit number is CGMCCNo.6733;
Described plant lactobacillus HM-10 deposit number is CGMCCNo.6741.
7. the preparation method of probiotics preparation according to claim 6, mixes stage by stage solid state fermentation and comprises that the seed culture of Bacillus licheniformis BL-09, subtilis HM-66 and plant lactobacillus HM-10 and solid state fermentation thereof cultivate, it is characterized in that,
Described seed culture medium mass percent consists of: peptone 1%, and glucose 2%, adds water to 100%, pH7.0-7.2;
Described solid-state fermentation culture medium mass ratio consists of: dregs of beans, wheat bran, Semen Maydis powder=10:9-10:0.5-1, water content is controlled at 60-70%.
8. the preparation method of probiotics preparation according to claim 6, described Bacillus licheniformis BL-09, subtilis HM-66 seed culture condition, be positioned on shaking table, and rotating speed is 150r/min, and temperature is 37 DEG C, cultivates 24h; Plant lactobacillus HM-10 seed culture condition, 37 DEG C leave standstill cultivation 24h;
Described solid state fermentation cultural method is: in solid-state fermentation culture medium, first inoculate Bacillus licheniformis BL-09 and subtilis HM-66 seed liquor, inoculum size is identical, be respectively 3.0-5.0%(v/w), controlled fermentation temperature 30-37 DEG C, humidity 70-80%, mixed bacterium is cultivated 12-24h, add plant lactobacillus HM-10 seed liquor, inoculum size is 1.0-3.0%(v/w again), other conditions are constant, the 12-24h that ferments again, the total time of fermenting is altogether 24-48h.
9. the preparation method of probiotics preparation according to claim 6, comprises the steps:
1. spawn culture
Bacillus licheniformis BL-09, subtilis HM-66 seed culture condition, be positioned on shaking table, and rotating speed is 150r/min, and temperature is 37 DEG C, cultivates 24h; Plant lactobacillus HM-10 seed culture condition, 37 DEG C leave standstill cultivation 24h;
2. solid state fermentation
In solid-state fermentation culture medium, first inoculate Bacillus licheniformis BL-09 and subtilis HM-66 seed liquor, inoculum size is respectively 3.0% (v/w), 37 ± 1 DEG C of culture temperature, humidity 75%, after fermentation 12h, inoculation 1.0% (v/w) plant lactobacillus HM-10 mixed fungus fermentation 36h;
3. dry
Fermentation ends obtains dry culture through fluidised bed drying.
10. the application of the probiotics preparation that prepared by the arbitrary described Bacillus licheniformis of claim 1-5, subtilis and plant lactobacillus in feed.
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| CN105707439A (en) * | 2016-03-17 | 2016-06-29 | 中国科学院合肥物质科学研究院 | Preparation method of animal health-care nutrient solution rich in probiotics |
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| US11297868B2 (en) | 2016-05-29 | 2022-04-12 | The State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization (Aro) (Volcani Center) | Method of generating bacterial compositions |
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| US11638432B2 (en) | 2017-07-12 | 2023-05-02 | Cj Cheiljedang Corporation | Feed additive comprising Bacillus subtilis and Bacillus licheniformis, a feed composition comprising the feed additive and a method for producing the feed additive |
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| CN109618552A (en) * | 2017-08-04 | 2019-04-12 | Cj第制糖株式会社 | The method of feed addictive comprising bacillus subtilis and bacillus licheniformis, the fodder compound comprising the feed addictive and the production feed addictive |
| CN107988117A (en) * | 2017-12-28 | 2018-05-04 | 内蒙古和美科盛生物技术有限公司 | A kind of compound probiotic decomposing agent and preparation method and application |
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| CN109971675A (en) * | 2019-03-22 | 2019-07-05 | 湖南碧野生物科技有限公司 | One lactobacillus plantarum SCUEC6 bacterial strain and its application |
| CN110250319A (en) * | 2019-07-10 | 2019-09-20 | 福州大学 | A kind of preparation of novel corynebacterium glutamicum waste residue probiotics |
| CN112625935A (en) * | 2020-09-25 | 2021-04-09 | 深圳合民生物科技有限公司 | MRS solid culture medium, microbial compound bacterium agent for inhibiting staphylococcus aureus and preparation method thereof |
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