A kind of additive for microbe feedstuff
Technical field: the invention belongs to feed additive field, particularly contain high-effective microorganism feedstuff additive product and production method and the application of multiple active bacteria formulation.
Background technology: at present China's milk cow amount of livestock on hand is approximately more than 1,000 ten thousand, more than 900 ten thousand tons of milk yields, the milk cattle cultivating industry develop the development that also promotes and promoted China's feed and additives industries rapidly; But China's milk cow forage still rests in the simple mixing of three aniseed (being corn, wheat bran, grouts), causes in formula that protein feeds is single, the amino acid mismatch, and the output of milk compares with American-European countries with quality that all there is a big difference.The research and development high-efficiency biological active fodder additives, for the level that improves China's milk cow production, improve the quality of milk, reduce the incidence of disease of milk cow gastrointestinal disease and mammitis, reduce the bad smell of fecaluria and the eliminating amount of fecaluria nitrogen, environmental contamination reduction and fully effective utilization of raising feed resource (namely improve the utilization rate of cheap roughage, improve the utilization rate of nutriment in feed etc.) have important actual application value.On the order field, popular active bacteria formulation is all single bacterium mostly, and be mainly used in monogastric animal feed, the single culture preparation of 2-3 family that only has at present the U.S. and France due to the product of ruminant (as milk cow) is in China's publicity and do cultivation and promote experiment.Bacillus subtilis, Lactobacillus plantarum are all the probios that can be applied to feed additive industry, and the various enzyme preparations that the probio metabolism produces and useful Metabolite thereof are promoting digestion, stablize the gut flora environment, having effect preferably aspect the raising efficiency of feed utilization; Aspergillus awamori can metabolism produce the plurality of enzymes preparation that comprises dextranase, and its culture can effectively improve the effect of digesting and assimilating of feed as feed addictive.Development has scientific matching, and successful contain multiple beneficial microorganism and the strong composite feed additive of enzyme activity for promoting aquaculture, particularly the milk cattle cultivating industry has very important significance.
Summary of the invention:
The technical problem that the present invention solves is to provide a kind of high-efficiency biological active fodder additives products, and another technical problem that the present invention solves is to provide high-efficiency biological active fodder additives products production method and application thereof.
Additive for microbe feedstuff consists of:
Aspergillus niger spore concentration 1.0~9.0 * 10
8Individual/gram, Trichoderma viride spore concentration 1.0~9.0 * 10
8Individual/gram, bacillus subtilis are 1.0~9.0 * 10
8Individual/gram, saccharomyces cerevisiae 1.0~8.5 * 10
8Individual/gram, Lactobacillus rhamnosus 1.3~8.5 * 10
8Individual/gram.
The concrete production method step of product of the present invention is as follows:
1. the cultivation of microbial bacterial agent preparation: training and drying obtain aspergillus niger spore, Trichoderma viride spore, bacillus subtilis, saccharomyces cerevisiae and Lactobacillus rhamnosus respectively.
(1) aspergillus niger spore preparation: slant pore suspension liquid is inoculated on 10 ° of Brix brewer's wort solid mediums, cultivates 2 days half to covering with spore for 30 ℃, and the low temperature fluidized bed drying is pulverized dry thing.
(2) Trichoderma viride spore preparation: slant pore suspension liquid is inoculated on 10 ° of Brix brewer's wort solid mediums, cultivates 2 days half to covering with spore for 30 ℃, and the low temperature fluidized bed drying is pulverized dry thing.
(3) preparation of bacillus subtilis: after the inclined-plane bacterial strain is activated, the access liquid seed culture medium, be seeded to fermentation tank after 37 ℃, the shaking table of 180rpm are cultivated 24h in, at 37 ℃, ventilating ratio 1: 1 (v/v), 400rpm cultivates 2 days to the logarithm later stage.After fermentation ends, add the filter aid precipitated calcium carbonate, through plate-frame filtering, fluidized bed drying.
(4) preparation of saccharomyces cerevisiae: slant strains access triangular flask, be cultured to logarithmic phase after switching enter in fermentation tank, controlling temperature is 28 ℃, ventilating ratio is 1: 1.5 (v/v), cultivates about 20 hours to logarithmic phase.After fermentation ends, add the filter aid precipitated calcium carbonate, through plate-frame filtering, fluidized bed drying.
(5) preparation of Lactobacillus rhamnosus: slant strains access triangular flask, be cultured to logarithmic phase after switching enter in fermentation tank, controlling temperature is 45 ℃, ventilating ratio is 1: 0.1 (v/v), cultivates about 18 hours to logarithmic phase.Fermenting obtains wet thallus complete the separation through plate-frame filtering, adds the protective agent that consists of 10% defatted milk, 5% trehalose, 5% glycerine, obtains microbial inoculum by freeze drying, and moisture is lower than 10%.
2, microbial inoculum is composite: above-mentioned various bacterium are proportionally carried out composite, the microbial inoculum compound proportion is as follows: aspergillus niger spore 15-25 part, Trichoderma viride spore 10-25 part, bacillus subtilis 10-25 part, saccharomyces cerevisiae 15-25 part and Lactobacillus rhamnosus 25-40 part.
The bacterial classification that the present invention adopts is as follows:
Aspergillus niger (Aspergillus niger) CGMC No.3.5269, Trichoderma viride (Trichoderma viride) CGMCC No.3.2942, bacillus subtilis (Bacillus subtilis) CGMCC No.1.210, saccharomyces cerevisiae (Saccharomyces cervisiac) CGMCC No.4429, Lactobacillus rhamnosus (Lactobacillus rhamnosus) CGMCC No.4430.
Saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.4429 bacterial classification is preservation of bacteria strain in the application of a strain osmophilic yeast bacterium with name of patent application, number of patent application 201110105966, and open day is 2011.09.14.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) CGMCC No.4430 bacterial strain characteristics are as follows: examine under a microscope, this bacterial strain is shaft-like, and width is less than 1 μ m, and 2 to 3 bacillus are easy to be linked to be and link together; On solid medium, this bacterium bacterium colony is milky, and smooth surface is moistening, thickness, and the edge is more neat.Compare with original bacterium, this mutagenic strain is significantly less than starting strain on form.Starting strain Lactobacillus rhamnosus CGMCCNo.1.2134 is purchased from Chinese microorganism strain preservation reason committee's common micro-organisms center.Lactobacillus rhamnosus of the present invention adopts following flow process to carry out seed selection: the original bacterial classification that sets out → test tube activation → high temperature acclimation → dithyl sulfate (DES) mutagenesis → high sugared plate screening → nitrosoguanidine (NTG) mutagenesis screening → high temperature bacterium screening → shaking flask is sieved → test of mitotic stability experiment → 5L fermentation tank again.Purpose bacterial strain CGMCC No.4430 is done the experiment of 5L lactic acid fermentation tank, and result shows: compare with starting strain, CGMCC No.4430 glucose-tolerant concentration can reach 270g/L, compares with original bacterium and has improved 95%; After fermentation ends, lactic acid content is 60g/L, compares with original bacterium and has improved 158%.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) CGMCC No.4430 depositary institution is Chinese common micro-organisms culture presevation administrative center.Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
In the present invention, aspergillus niger spore, Trichoderma viride spore adopt blood cell plate counting method, and other bacterial classification adopts microorganism plate count method to detect viable count.
What in the present invention, microbial inoculum was composite than the ratio of greater inequality example is: aspergillus niger spore 15-20 part, Trichoderma viride spore 10-20 part, bacillus subtilis 10-20 part, saccharomyces cerevisiae 15-20 part and Lactobacillus rhamnosus 25-35 part.
The invention product adopts the microbial inoculum compound proportion technology of scientific research effectively to guarantee reasonable constituents and the ratio of various microbial bacterial agents in product, makes the enzyme of microorganisms in product can effectively promote digesting and assimilating of feed in Cow-feeding; Compounded technology makes product significantly improve at the digestibility of milk cow forage, and the yield ratio of unit consumption feed significantly improves than contrasting to have had, and the output of milk cow and quality are improved and ensure.
Safety is used in the stable performance of invention product, with other feed addictive all without incompatibility.Product of the present invention is stable and controllable for quality, non-environmental-pollution, and appearance can be put forward immunity can improve culture benefit.Product meets the requirement of green feed additive fully.The order that is significantly improved weightening finish and feed conversion rate effect.
In product of the present invention, Lactobacillus rhamnosus and saccharomyces cerevisiae have booster action to the digestion of animal gastrointestinal tract, saccharomyces cerevisiae for the growth and breeding of rumen microorganism provides useful ammonia at acid, B family vitamin and other trace element, increases the synthetic quantity of rumen microorganism egg matter in cud.Lactobacillus rhamnosus discharges at the small intestine of milk cow, can consume the oxygen in animal intestinal, pathogenic bacteria such as aerobic bacteria (being mainly Escherichia coli, salmonella etc.) harmful in enteron aisle are suppressed, can also suppress the synthetic of ammonia and amine, benefit strengthens immunity, reach and improve milk cow intestines and stomach microbial ecological balance, be conducive to purpose healthy and the raising production performance; Cellulase, dextranase, lignin decomposition enzyme and the amylase of the effective dose that bacillus subtilis contains in enzyme that the enteron aisle metabolism produces and aspergillus niger, Trichoderma Viride culture is conducive to digesting and assimilating of Fibrous feedstuff in the ruminant feed such as milk cow, improve the microecological balance of intestine of ruminants, improve efficiency of feed utilization, improve milk production of cow, improve the quality of milk, improve the milk cow body immunity, reduce intestines problem, purify feeding environment.This feed addictive can be applicable to the milk cattle cultivating industry, can significantly improve milk crop and quality.
Product of the present invention can improve the milk cow body immunity, reduces the incidence of disease of recessive mastitis, intestines problem.At present, in China milk cows, the recessive mastitis incidence of disease is higher, in a single day milk cow suffers from mammitis just must the injection of antibiotics treatment, and the milk of having injected institute's output within antibiotic milk cow 7 days all can not be sold (in milk can residual antibiotic human body is had harm).Use product of the present invention can reduce the cow subclinical mastitis incidence of disease, feeding experiment shows that product of the present invention reduces the mammitis incidence of disease 70% (comparing with control group), obviously reduce the generation of mastitis for milk cows, reduce the antibiotic output of using and containing antibiotic milk, indirectly improved culturist's benefit.
The level that product of the present invention is produced improving China milk cow, the aspects such as effective utilization (namely improve the utilization rate of cheap phase feed, improve the utilization rate of nutriment in feed etc.) that fully improve feed resource all have very high value.
The present invention also is fit to feeding of other ruminants such as beef cattle, sheep.
The specific embodiment:
The following examples can make the present invention of those skilled in the art comprehend, but do not limit the present invention in any way.
Embodiment 1: high-efficiency activated feedstuff additive product
Product is composed as follows: aspergillus niger spore concentration 1.0~3.0 * 10
8Individual/gram, Trichoderma viride spore concentration 1.0~3.0 * 10
8Individual/gram, bacillus subtilis are 1.0~3.0 * 10
8Individual/gram, saccharomyces cerevisiae 1.0~3.5 * 10
8Individual/gram, Lactobacillus rhamnosus 1.3~4.5 * 10
8Individual/gram.
Microbial inoculum is composite: carry out composite according to following ratio above-mentioned various microbial inoculums:
15 parts of aspergillus nigers, green this 15 parts, mould spore, 15 parts of bacillus subtilises, 25 parts of saccharomyces cerevisiaes, 30 parts of Lactobacillus rhamnosus.
Embodiment 2: high-efficiency activated feedstuff additive product
Product is composed as follows: aspergillus niger spore concentration 5 * 10
8Individual/gram, Trichoderma viride spore concentration 5.0 * 10
8Individual/gram, bacillus subtilis are 5 * 10
8Individual/gram, saccharomyces cerevisiae 6 * 10
8Individual/gram, Lactobacillus rhamnosus 6 * 10
8Individual/gram.
Microbial inoculum is composite: carry out composite according to following ratio above-mentioned various microbial inoculums:
Aspergillus niger 20 valencys, 10 parts, Trichoderma viride spore, 20 parts of bacillus subtilises, 18 parts of saccharomyces cerevisiaes, 32 parts of Lactobacillus rhamnosus.
Embodiment 3: high-efficiency activated feedstuff additive product production method described in example 1 and 2
Product processes mainly comprises production and the complex process of microbial bacterial agent.
The production of microbial bacterial agent:
The preparation method of aspergillus niger and Trichoderma viride spore
1. inclined-plane cultural method
Slant medium 10mL adds test tube, and 121 ℃, after the 20min sterilization, put the inclined-plane, cooling, inoculation.30 ℃ of cultivations are paved with the inclined-plane to the black spore.
2.K formula blake bottle spore
Get 10 ° of Brix brewer's worts and add 2% agar, the 500mL K formula of packing into blake bottle, 121 ℃, after the 20min sterilization, the paving inclined-plane is cooling.Access spore suspension 1mL guarantees that suspension is inoculated in whole media surface; Be sidelong into insulating box, 30 ℃ of cultivations are paved with the inclined-plane to the black spore.
3. solid-state amplification is cultivated
K formula phialosporae is made spore suspension, and spore concentration is greater than 1.0 * 10
8Individual/mL.Get 200kg solid medium (wheat bran 140kg, 10 ° of Brix brewer's wort 60L), fully put into tray after mixing, sterilization is 1 hour under 121 ℃.After cooling, the access spore suspension stirs.Cultivation temperature is controlled at 30 ℃, humidity 80-90%, every 10 hours stirrings once, incubation time 3 days; Treat that compost covers with spore and can finish to cultivate
Drying and crushing: after fermentation ends, tray is placed on fluidized bed drying, baking temperature is controlled at 60 ℃, when moisture content of material will be following lower than 10%, with pulverizer, solid culture medium is carried out powder sulphur, and the crushing material aperture is more than 100 orders.
The preparation method of bacillus subtilis
Culture medium forms (g/L): glucose 60, yeast extract 20, dipotassium hydrogen phosphate 0.5, epsom salt 0.5, pH6.8.
(1) first order seed is cultivated: 500mL shaking flask liquid amount is 100mL, access one ring bacillus subtilis after sterilization, and on shaking table, 180 rev/mins, 37 ℃ of cultivation temperature, incubation time 24 hours is to logarithmic phase;
(2) secondary seed is cultivated: in the seeding tank of first order seed according to the inoculum concentration access 30L of 10% (v/v), and liquid amount 21L, 37 ℃ of cultivation temperature, 200 rev/mins of mixing speeds, ventilation (V/V) 1: 0.5, tank pressure 0.05Mpa, incubation time 24 hours is to logarithmic phase.
(3) fermentation tank culture: the seed in seeding tank is accessed the 300L fermentation tank with 10% (v/v) inoculum concentration, liquid amount 210L, 37 ℃ of cultivation temperature, mixing speed 200 turns minute, ventilation (V/V) 1: 0.5. tank pressure 0.05Mpa, incubation time 24 hours is cultivated and is finished bacteria concentration 5.0 * 10
9Individual/mL.
(4) centrifugation: adopt plate-frame filtering to separate and obtain thalline.
(7) vacuum freeze drying: adopt drying process with atomizing to process the acquisition dry bacteria after adding protective agent; Protective agent consists of defatted milk 10%, lactose 5%, glycerine 5%.
The preparation method of S. cervisiae:
From inclined-plane switching saccharomycete to liquid shaking bottle, liquid shaking bottle through three grades spread cultivation after switching enter fermentation tank and enlarge cultivation, cultivate that complete low temperature is concentrated, carrier mixes, liquid bed drying process acquisition active dry yeasr microbial inoculum.
The cultivation of yeast cells: adopt the slant strains acquisition yeast cells that spreads cultivation step by step;
(1) first order seed is cultivated: in yeast slant strains access 500mL shaking flask, culture medium loading amount 100mL, rotary shaking table 120 turn minute, 30 ℃ of cultivation temperature, incubation time 18 hours;
(2) secondary seed is cultivated: according in 10% inoculum concentration access 500mL secondary seed shaking flask, condition of culture is identical with first order seed with first order seed;
(3) three grades of seed culture: with in three grades of seed shaking flasks of 10% inoculum concentration access 5000mL, culture medium loading amount 1000mL, rotary shaking table 100 turn minute, 30 ℃ of cultivation temperature, incubation time 18 hours with secondary seed;
(4) first class seed pot is cultivated: with the first class seed pot of three grades of seeds take 5% inoculum concentration access total measurement (volume) as 150L, fermentation medium loading amount 100L, 30 ℃ of cultivation temperature, 100 rev/mins of mixing speeds, ventilation (V/V) 1: 0.5, tank pressure 0.05Mpa, incubation time 16 hours;
(5) fermentation tank culture: the first class seed pot bacterial classification is accessed total measurement (volume) as 3 tons of secondary seed tanks take 5% inoculum concentration (v/v), 2 tons of fermentation medium loading amounts, 30 ℃ of condition of culture cultivation temperature, 100 rev/mins of mixing speeds, ventilation in early stage (V/V) 1: 05, tank pressure 0.05Mpa, early stage, incubation time was 12 hours; Later stage tank pressure 0.05Mpa, mixing speed 50 turns minute, incubation time 8 hours, the yeast concentration of cultivating latter stage reaches 3.0 * 10
9Individual/mL.
(6) concentrated: zymotic fluid is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains the saccharomycete concentrate.
(7) add carrier: add the carrier that mixes in the saccharomycete concentrate, mix; The weight ratio of concentrate and carrier is 0.5-0.7: 1, and vehicle group becomes: CaCO
340 parts, 20 parts, dextrin, 20 parts of corn protein powders.
(8) drying: fluidized bed drying, 50 ℃ of baking temperatures.
Culture medium adopts 10% malt extract medium or adopts the molasses culture medium of adding the inorganic salts such as ammonium sulfate, specifically cultivates production technology referring to Xiao Dongguang " production of Active Dry Yeast and application technology ".
The preparation of Lactobacillus rhamnosus microbial inoculum:
Slant strains access triangular flask, be cultured to logarithmic phase after switching enter in fermentation tank, controlling temperature is 45 ℃, ventilating ratio is 1: 0.1 (v/v), cultivates about 18 hours to logarithmic phase.Fermenting obtains wet thallus complete the separation through plate-frame filtering, adds the protective agent that consists of 10% defatted milk, 5% trehalose, 5% glycerine, obtains microbial inoculum by freeze drying, and moisture is lower than 10%.
The experiment of product effect:
Selection and the experimental design of test ox: tested 7 days-October 7 September in 2010 and carry out in Wuzhong City kumquat cattle farm, Ningxia, the random pair principle is taked in this experiment, with 40 oxen random be divided into test group and control group, every group of 20 oxen, add product 40g by every every day in daily ration, fed 60 days, result shows experimental group daily gain 972.7g, control group daily gain 746.8g, difference is (P<0.01) extremely significantly.After feeding 5 days, its ight soil dry with feed before compare, reduce 30%.
Use high-efficiency biological active fodder additives after 1 month, find that milk crop is more stable, (group of not feeding declines by a big margin, because the test ox has spent peak of lactation), butterfat percnetage has also increased, and effect is fine.From the milk cow appearance, the hair of the milk cow of hello high-efficiency biological active fodder additives is bright, and appetite is good, and the mammitis incidence of disease reduces.