CN103952336A - Bacillus licheniformis-Bacillus subtilis-Lactobacillus casei preparation and preparation method thereof - Google Patents

Bacillus licheniformis-Bacillus subtilis-Lactobacillus casei preparation and preparation method thereof Download PDF

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CN103952336A
CN103952336A CN201410121427.7A CN201410121427A CN103952336A CN 103952336 A CN103952336 A CN 103952336A CN 201410121427 A CN201410121427 A CN 201410121427A CN 103952336 A CN103952336 A CN 103952336A
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beans
dregs
wheat bran
bacillus licheniformis
semen maydis
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CN103952336B (en
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高鹏飞
赵树平
姚国强
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INNER MONGOLIA SCI-PLUS BIOTECH Co Ltd
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INNER MONGOLIA SCI-PLUS BIOTECH Co Ltd
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    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Abstract

The present invention discloses a Bacillus licheniformis-Bacillus subtilis-Lactobacillus casei composite probiotic preparation and a preparation method thereof, and belongs to the technical field of feed microorganisms, wherein soybean meal, wheat bran and corn powder are adopted as matrixes, and Bacillus licheniformis-Bacillus subtilis-Lactobacillus casei are adopted to carry out solid state fermentation to obtain the probiotic preparation. According to the present invention, the preparation method has characteristics of low production cost, economic benefit increase and simple operation, and can be widely used for the probiotic solid state fermentation technology in the microecology field; the bacterial mixing solid state fermentation is adopted, and the mutual-benefit symbiotic synergism effect among the strains is adopted, such that the yield and the bacterial concentration can be increased, the number of live bacteria in the obtained fermented product is high, and the proteins in the soybean meal and the corn powder are easily decomposed into small peptides and free amino acids so as to be easily digested and absorbed by intestine and stomach; and when the product is added to a feed, the feed utilization efficiency can be effectively increased by 13-25% when the product is fed to milk cow.

Description

Bacillus licheniformis, subtilis and lactobacterium casei preparation and preparation
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, taking probiotic bacterium Bacillus licheniformis BL-09(Bacillus licheniformis BL-09), subtilis HM-66(Bacillus subtilis HM-66) and lactobacterium casei HM-09(Lactobacillus casei HM-09) as the solid-state fermentation technology of bacterial classification, this technology can be widely used in the aspects such as animal microecological.
Background technology
At present, probiotic composition adopts probiotic bacterium directly and liquid cow's milk and soya-bean milk fermentation more, obtains functional yogurt milk or soybean milk yoghurt, and all the other adopt liquid state fermentation, directly collect probiotic bacterium thalline, are 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.Therefore the application adopts Bacillus licheniformis BL-09, subtilis HM-66 and lactobacterium casei HM-09 mixed solid fermentation dregs of beans, wheat bran and Semen Maydis powder, adopt solid state fermentation, 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 lactobacterium casei HM-09 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.
Chinese invention patent 200810175856.7 discloses a strain and has had the high density fermentation technology of the lactobacterium casei (LACTOBACILLUS CASEI ZHANG) of prebiotic effect, comprise proliferated culture medium and optimization of fermentation condition control, and the technology of preparing of the high vigor freeze-dried vaccine powder of this bacterial strain.The freeze-dried vaccine powder of the lactobacterium casei of the above viable count of 1011CFU/ML can be obtained by the present invention, preparation and the application of this bacterial strain probiotics leaven and probiotics preparation can be directly applied to.
Name be called a kind of lactobacterium casei and the application Chinese invention patent in solid state fermentation thereof, application number is 200710118485.4, this disclosure of the invention a kind of lactobacterium casei and the application in solid state fermentation thereof.Described lactobacterium casei (LACTOBACILLUS CASEI ZHANG) is the probiotic bacterium that separates the acidproof and resistance to bile acide obtaining from koumiss; This bacteria strain has been preserved in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number: CGMCC NO.1697.Lactobacterium casei of the present invention (LACTOBACILLUS CASEI ZHANG) can be realized the application aspect solid state fermentation, the particularly application in milk-product, heath food, microbiological feed and animal health goods as starter; The method that lactobacterium casei thalline survival rate of the present invention improves, is a kind of method of optimizing solid medium, can obtain higher cell density and thalline survival rate; Lactobacterium casei solid state fermentation viable count of the present invention is liquid MRS fermentation the more than 20 times of viable count.
Name is called the lactobacterium casei for fodder additives probiotic bacterium solid fermentation, animal bifidobacteria, the patent of invention of plant lactobacillus and subtilis, application number is 01310321977.9, this disclosure of the invention is for the lactobacterium casei of fodder additives probiotic bacterium solid fermentation, animal bifidobacteria, plant lactobacillus and subtilis, it is characterized in that: described subtilis HM-66, lactobacterium casei F-08, plant lactobacillus HM-20 and animal bifidobacteria V9 are probiotic bacterium, be preserved in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number is respectively CGMCCNo.6733, CGMCCNo.6735, CGMCCNo.6744 and CGMCCNo.5470, subtilis HM-66, the preservation date of lactobacterium casei F-08 and plant lactobacillus 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 method.
For the animal bifidobacteria of fodder additives probiotic bacterium solid fermentation and the Chinese invention patent of subtilis, application number is 201310321979.8, and invention relates to a kind of producing and has the fermented type dregs of beans of special sour fragrance and the probiotic bacterium solid fermentation that can be used for food and feed additive field containing high viable count and high protein degradation rate.Subtilis HM-66 and animal bifidobacteria V9 for solid fermentation are probiotic bacterium, are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is respectively CGMCCNo.6733 and CGMCCNo.5470.
Be used for plant lactobacillus and the careful Chinese invention patent of subtilis of the probiotic bacterium solid fermentation of feed additive field, application number is 201310322177.9, it is little that a kind of pollution is disclosed, easy to operate, be conducive to plant lactobacillus and subtilis for fodder additives probiotic bacterium solid fermentation that stomach is digested and assimilated, it is characterized in that: described subtilis HM-66 and plant lactobacillus HM-20 are probiotic bacterium, be preserved in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number is respectively CGMCCNo.6733 and CGMCCNo.6744.
The lactobacterium casei and the careful Chinese invention patent of subtilis that are used for the probiotic bacterium solid fermentation of feed additive field, application number is 201310322953.5.Disclose a kind of pollute little, easy to operate, be conducive to subtilis and lactobacterium casei for fodder additives probiotic bacterium solid fermentation that stomach is digested and assimilated, it is characterized in that: described subtilis HM-66) and lactobacterium casei F-08 be probiotic bacterium, be preserved in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number is respectively CGMCCNo.6733 and CGMCCNo.6735.
Name is called a kind of effective prevention and improves the compound lactobacillus probiotics Chinese patent of garget, application number is 201310094615.0, this patent discloses a kind of compound micro-ecological preparation that prevents and improve garget, lactobacterium casei HM-09 in compound micro-ecological preparation (L.casei HM-09) mass fraction is 10%, plant lactobacillus HM-10(L.plantarum HM-10) mass fraction is 10%, all the other 80% are zeolite powder, L.casei HM-09 number of viable >=2.5 × 108cfu/g in compound micro-ecological preparation simultaneously, L.plantarum HM-10 number of viable >=2.5 × 108cfu/g, described lactobacterium casei and plant lactobacillus have been great Tun road, Beijing in address on October 29th, 2012, the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, lactobacterium casei HM-09 (L.casei HM-09) preserving number: CGMCC No.6736, plant lactobacillus HM-10(L.plantarum HM-10) preserving number: CGMCC No.6741.
Summary of the invention
The object of the invention is to adopt Bacillus licheniformis BL-09, subtilis HM-66 and lactobacterium casei HM-09 to carry out solid state fermentation dregs of beans, wheat bran and Semen Maydis powder, a kind of probiotic bacterium solid-fermented technique that can be widely used in micro-ecosphere taking dregs of beans, wheat bran and Semen Maydis powder as matrix is provided, the viable count of this method leavened prod is high, and the degradation rate of dregs of beans protein is high.This preparation method's 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 probiotic bacterium solid-fermented technique that can be widely used in micro-ecosphere taking dregs of beans, wheat bran and Semen Maydis powder as matrix, the cultivation and the solid state fermentation thereof that comprise Bacillus licheniformis BL-09, subtilis HM-66 and lactobacterium casei HM-09 are cultivated, and obtain solid state fermentation dregs of beans, wheat bran and the Semen Maydis powder product of probiotic bacterium.
Described Bacillus licheniformis BL-09, subtilis HM-66 and lactobacterium casei HM-09 seed culture medium are: peptone 1-2%, glucose 2-4%, adds water to 100%, pH7.0-7.2.
Described Bacillus licheniformis BL-09, 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; Lactobacterium casei HM-09 seed culture condition, 30-37 DEG C leaves standstill cultivation 20-24h.
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 respectively 2-8% (v/w), controlled fermentation temperature 30-38 DEG C, humidity 40-65%, mixed bacterium is cultivated 12-18h; Add lactobacterium casei HM-09 seed liquor, inoculum size is 1-8% (v/w) again, and other conditions are constant, then the 12-36h that ferments.Dry acquisition after fermentation ends.
Solid-state fermentation culture medium consists of dregs of beans, wheat bran and Semen Maydis powder, and the composition of described dregs of beans, wheat bran and Semen Maydis powder and mass ratio ratio are: dregs of beans, wheat bran, Semen Maydis powder=10:8-10:0.5-1.Water content is controlled at 40-75%.
Bacillus licheniformis BL-09, subtilis HM-66 and lactobacterium casei HM-09 mixed fungus fermentation, after fermentation ends, free ammonical nitrogen content reaches 1109.1umol/g, before fermentation, improve approximately 11 times, Bacillus licheniformis BL-09 and subtilis HM-66 gemma rate are fermented more separately and are improved respectively 2.1 times and 1.5 times.
Subtilis HM-66 and lactobacterium casei HM-09 were in preservation on October 29 in 2012, and preserving number is respectively CGMCC No.6733 and CGMCC No.6736.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 is respectively (Bacillus licheniformis) BL-09, Bacillus licheniformis BL-09 and is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 31st, 2011, bacterium preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode 100101; Preserving number is CGMCC No.5686.
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, 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 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, 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, 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 resistance to light salt.
Beneficial effect:
In the present invention, adopt Bacillus licheniformis BL-09, subtilis HM-66 and lactobacterium casei HM-09 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 lactobacterium casei HM-09 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, breed entering after 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.Lactobacterium casei 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 lactobacillus casei fermented milk produces are inhibited to Angiotensin, and then play hypotensive activity; Often take lactobacterium casei active bacteria formulation, can reduce the cholesterol in blood.Subtilis, Bacillus licheniformis and lactobacterium casei composite probiotics preparations that the present invention obtains, with above function, have good application development prospect.Make in feed, to add while use in product of the present invention, this series products can effectively improve the utilising efficiency 13-25% of feed while being used for Cow-feeding.The addition of product of the present invention in feed is 20-50 gram/kg.
After product of the present invention uses, the conception rate of milk cow has improved more than 12%, can improve as calculated the year milk yield more than 210 kilograms of milk cow.
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.
1. the seed liquor of thalline preparation
Bacillus licheniformis BL-09, subtilis HM-66 and lactobacterium casei HM-09 receive in the substratum that liquid seeds is housed from the glycerine pipe of cold storage, Bacillus licheniformis BL-09, subtilis HM-66 seed culture condition, be positioned on shaking table, rotating speed is 150r/min, temperature is 37 DEG C, cultivates 24h; Lactobacterium casei HM-09 seed culture condition, 37 DEG C leave standstill cultivation 24h.
Wherein liquid seed culture medium (g/L) is: peptone 1%, glucose 2%, adds water to 100%, pH7.0-7.2.
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 5% (v/w), 35 DEG C of controlled fermentation temperature, and humidity 55%, mixed bacterium is cultivated 12h; Add lactobacterium casei HM-09 seed liquor, inoculum size is 4% (v/w) again, 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: dregs of beans, wheat bran, Semen Maydis powder=10:8:0.5.40%, 115 DEG C of autoclaving 25min of moisture content.
3. 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 4.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.
5. 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.
Below the determining of some parameters of the production method to the probiotics fermention product taking dregs of beans as matrix, and the optimization of some culture condition.
Embodiment 1:
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% (v/w), 35 DEG C of controlled fermentation temperature, and humidity 60%, mixed bacterium is cultivated 12h; Add lactobacterium casei HM-09 seed liquor, inoculum size is 5% (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 composition of described dregs of beans, wheat bran and Semen Maydis powder and mass ratio ratio are: dregs of beans, wheat bran, Semen Maydis powder=10:10:0.5, water content is controlled at 60%.
Recording lactobacterium casei HM-66 viable count is 4.05 × 10 9cfu/g, HM-09 subtilis HM-66 viable count is 1.05 × 10 10cfu/g, Bacillus licheniformis BL-09 viable count is 1.29 × 10 10cfu/g, after fermentation ends, free ammonical nitrogen content reaches 1038.7umol/g.
Embodiment 2:
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% (v/w), 37 DEG C of controlled fermentation temperature, and humidity 40%, mixed bacterium is cultivated 15h; Add lactobacterium casei HM-09 seed liquor, inoculum size is 5% (v/w) again, 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 dregs of beans, wheat bran and Semen Maydis powder, and the composition of described dregs of beans, wheat bran and Semen Maydis powder and mass ratio ratio are: dregs of beans, wheat bran, Semen Maydis powder=10:8:1, water content is controlled at 70%.
Recording lactobacterium casei HM-09 viable count is 4.09 × 10 9cfu/g, subtilis HM-66 viable count is 0.95 × 10 10cfu/g, Bacillus licheniformis BL-09 viable count is 1.18 × 10 10cfu/g, after fermentation ends, free ammonical nitrogen content reaches 1019.9umol/g.
Embodiment 3:
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% (v/w), 30 DEG C of controlled fermentation temperature, and humidity 65%, mixed bacterium is cultivated 18h; Add lactobacterium casei HM-09 seed liquor, inoculum size is 5% (v/w) again, 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 dregs of beans, wheat bran and Semen Maydis powder, and the composition of described dregs of beans, wheat bran and Semen Maydis powder and mass ratio ratio are: dregs of beans, wheat bran, Semen Maydis powder=10:9:0.8, water content is controlled at 70%.
Recording lactobacterium casei HM-09 viable count is 4.10 × 10 9cfu/g, 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
When Bacillus licheniformis BL-09, subtilis HM-66 and lactobacterium casei HM-09 inoculum size are respectively 3.0% (v/w), 3.0% (v/w) and 1.0% (v/w), and first utilize after Bacillus licheniformis BL-09 and subtilis HM-66 mixed fungus fermentation 12h, again with lactobacterium casei HM-09 mixed fungus fermentation 36h, 37 ± 1 DEG C of culture temperature, amount to incubation time 48h, its viable count is higher, and recording lactobacterium casei HM-09 viable count is 4.55 × 10 9cfu/g, subtilis HM-66 viable count is 1.35 × 10 10cfu/g, Bacillus licheniformis BL-09 viable count is 1.55 × 10 10cfu/g, and now the content of free ammonical nitrogen is the highest, reaches 1109.1umol/g.

Claims (7)

1. a composite probiotics preparations, can be made by following method: in solid-state fermentation culture medium, first inoculate Bacillus licheniformis BL-09 and subtilis HM-66 seed liquor, inoculum size is respectively 2-8% (v/w), controlled fermentation temperature 30-38 DEG C, humidity 40-65%, mixed bacterium is cultivated 12-18h; Add lactobacterium casei HM-09 seed liquor, inoculum size is 1-8% (v/w) again, and other conditions are constant, then the 12-36h that ferments, and fermentation ends is dry.
2. composite probiotics preparations according to claim 1: it is characterized in that, solid-state fermentation culture medium consists of dregs of beans, wheat bran and Semen Maydis powder, and the composition of described dregs of beans, wheat bran and Semen Maydis powder and mass ratio ratio are: dregs of beans, wheat bran, Semen Maydis powder=10:8-10:0.5-1.
3. composite probiotics preparations according to claim 1: it is characterized in that, Bacillus licheniformis (Bacillus licheniformis), preserving number is CGMCC No.5686.
4. the preparation method of composite probiotics preparations according to claim 1, comprise the steps: first to inoculate in solid-state fermentation culture medium Bacillus licheniformis BL-09 and subtilis HM-66 seed liquor, inoculum size is respectively 2-8%, controlled fermentation temperature 30-38 DEG C, humidity 40-65%, mixed bacterium is cultivated 12-18h; Add lactobacterium casei HM-09 seed liquor, inoculum size is 1-8% again, and other conditions are constant, then the 12-36h that ferments, and fermentation ends is dry; Solid-state fermentation culture medium consists of dregs of beans, wheat bran and Semen Maydis powder, and the composition of described dregs of beans, wheat bran and Semen Maydis powder and mass ratio ratio are: dregs of beans, wheat bran, Semen Maydis powder=10:8-10:0.5-1.Bacillus licheniformis, preserving number is CGMCC No.5686.
5. the preparation method of composite probiotics preparations according to claim 4, in solid-state fermentation culture medium, first inoculate Bacillus licheniformis BL-09 and subtilis HM-66 seed liquor, inoculum size is respectively 3%, 35 DEG C of controlled fermentation temperature, humidity 60%, mixed bacterium is cultivated 12h; Add lactobacterium casei HM-09 seed liquor, inoculum size is 5% 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 composition of described dregs of beans, wheat bran and Semen Maydis powder and mass ratio ratio are: dregs of beans, wheat bran, Semen Maydis powder=10:10:0.5, and water content is controlled at 60%; After fermentation ends, free ammonical nitrogen content reaches 1038.7umol/g.
6. the preparation method of composite probiotics preparations according to claim 4, 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%, 37 DEG C of controlled fermentation temperature, humidity 45%, mixed bacterium is cultivated 15h; Add lactobacterium casei HM-09 seed liquor, inoculum size is 5% again, 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 dregs of beans, wheat bran and Semen Maydis powder, the composition of described dregs of beans, wheat bran and Semen Maydis powder and mass ratio ratio are: dregs of beans, wheat bran, Semen Maydis powder=10:8:1, water content is controlled at 70%, and free ammonical nitrogen content reaches 1019.9umol/g.
7. the application of composite probiotics preparations according to claim 4 in livestock and poultry cultivation.
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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
CN114766597A (en) * 2022-03-24 2022-07-22 焦作市佰役安生物工程有限公司 Method for preparing antibacterial peptide feed additive by duplex mixed fermentation
CN114766597B (en) * 2022-03-24 2024-04-05 焦作市佰役安生物工程有限公司 Method for preparing antibacterial peptide feed additive through duplex mixed fermentation

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