CN102835564B - Biological feed additive - Google Patents
Biological feed additive Download PDFInfo
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- CN102835564B CN102835564B CN201110419570.0A CN201110419570A CN102835564B CN 102835564 B CN102835564 B CN 102835564B CN 201110419570 A CN201110419570 A CN 201110419570A CN 102835564 B CN102835564 B CN 102835564B
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- saccharomyces cerevisiae
- lactobacillus rhamnosus
- bacillus licheniformis
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- 239000003674 animal food additive Substances 0.000 title abstract description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 36
- 241000218588 Lactobacillus rhamnosus Species 0.000 claims abstract description 27
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 23
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 23
- 239000000654 additive Substances 0.000 claims description 16
- 150000001875 compounds Chemical class 0.000 claims description 5
- 235000013336 milk Nutrition 0.000 abstract description 33
- 239000008267 milk Substances 0.000 abstract description 33
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- 238000002360 preparation method Methods 0.000 abstract description 12
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- 238000002474 experimental method Methods 0.000 description 4
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
Belonging to the field of feed additives, the invention discloses a biological feed additive, and solves the technical problems of high efficiency biological feed additive products containing a plurality of live bacterium preparations, their production methods and application. The involved biological feed additive comprises: Aspergillus niger spores with a concentration of 1.0-9.0*10<8>/g, Bacillus licheniformis with a concentration of 1.0-9.0*10<8>/g, Saccharomyces cerevisiae with a concentration of 1.0-8.5*10<8>/g, and Lactobacillus rhamnosus with a concentration of 1.3-8.5*10<8>/g. The feed contains a variety of beneficial microorganisms and has strong enzyme activity. Tests prove that the feed can substantially improve the milk yield of diary cows and the milk components.
Description
Technical field: the invention belongs to feed additive field, particularly contain highly effective biological feed additive product and production method and the application of multiple active bacteria formulation.
Background technology: at present approximately more than 1,000 ten thousand of China's milk cow amount of livestock on hands, more than 900 ten thousand tons of milk yields, milk cattle cultivating industry develop the development that also promotes and promoted China's feed and additives industries rapidly; But China's milk cow feed still rests in the simple mixing of three aniseed (being corn, wheat bran, grouts), cause in formula that protein feed is single, amino acid mismatch, all there is a big difference compared with American-European countries with quality for the output of milk.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 sickness rate of milk cow gastrointestinal illness and mastitis, reduce the unpleasant odor of fecaluria and the eliminating amount of fecaluria nitrogen, the effective utilization (improve the utilization ratio of cheap roughage, improve the utilization ratio of nutritive substance in feed etc.) that reduces environmental pollution and abundant raising feed resource has important actual application value.Popular active bacteria formulation is all single bacterium mostly in the market, and be mainly used in monogastric animal feed, the single culture preparation that only has at present the U.S. and French 2-3 family for the product of ruminating animal (as milk cow) is in China's publicity and do cultivation and promote experiment.Subtilis, plant lactobacillus are all the probiotic bacteriums that can be applied to feed additive industry, the various zymins that probiotic bacterium metabolism produces and useful Metabolite thereof at promoting digestion, stablize intestinal microflora environment, raising has good effect aspect efficiency of feed utilization; Aspergillus awamori can metabolism produce the plurality of enzymes preparation including dextranase, and its culture can effectively improve the effect of digesting and assimilating of feed as fodder additives.Development has scientific matching, and successful contain multiple beneficial microorganism and the strong composite feed additive of enzyme activity for promoting aquaculture, particularly 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.
Biology feed additive consists of: aspergillus niger spore concentration 1.0～9.0 × 10
8individual/gram, Bacillus licheniformis is 1.0～9.0 × 10
8individual/gram, yeast 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 microbiobacterial agent preparation: spreading cultivation respectively and being dried obtains aspergillus niger spore, Bacillus licheniformis, yeast saccharomyces cerevisiae and lactobacillus rhamnosus.
(1) aspergillus niger spore preparation: slant pore suspension liquid is inoculated on 10 ° of Brix wort solid mediums, cultivates 2 days half to covering with spore for 30 ℃, and low temperature fluidized-bed dry, pulverize dry thing.
(2) preparation of Bacillus licheniformis: after inclined-plane bacterial strain is activated, access liquid seed culture medium, be seeded in fermentor tank after cultivating 24h at 37 ℃, the shaking table of 180rpm, at 37 ℃, ventilating ratio 1: 1 (v/v), 400rpm cultivates 2 days to the logarithm later stage.After fermentation ends, add flocculating aids light calcium carbonate, through Plate Filtration, fluidised bed drying.
(3) preparation of yeast saccharomyces cerevisiae: slant strains access triangular flask, transfer in fermentor tank after being cultured to logarithmic phase, controlling temperature is 28 ℃, ventilating ratio is 1: 1.5 (v/v), cultivates about 20 hours to logarithmic phase.After fermentation ends, add flocculating aids light calcium carbonate, through Plate Filtration, fluidised bed drying.
(4) preparation of lactobacillus rhamnosus: slant strains access triangular flask, transfer in fermentor tank after being cultured to logarithmic phase, controlling temperature is 45 ℃, ventilating ratio is 1: 0.1 (v/v), cultivates about 18 hours to logarithmic phase.Ferment complete through Plate Filtration separate obtain wet thallus, add the protective material that consists of 10% skimmed milk, 5% trehalose, 5% glycerine, obtain microbial inoculum by lyophilize, moisture content is lower than 10%.
2, microbial inoculum is composite: above-mentioned various bacterium are proportionally carried out composite, microbial inoculum compound proportion is as follows: aspergillus niger 25-50 part, Bacillus licheniformis 10-25 part, yeast saccharomyces cerevisiae 15-25 part, lactobacillus rhamnosus 25-40 part.
The bacterial classification that the present invention adopts is as follows:
Aspergillus niger (Aspergillus niger) CGMCC No.3.5269, Bacillus licheniformis (Bacillus licheniformis) CGMCC No.1.813, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCCNo.4429, lactobacillus rhamnosus (Lactobacillus rhamnosus) CGMCC No.4430.
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.4429 feature is as follows: examine under a microscope, the cell of this bacterial strain is avette, one end budding, and size is about 1 × 5 μ m; On solid medium, this bacterium bacterium colony is oyster white, and smooth surface is moistening, thickness, and edge is more neat and medium bigger than normal.Compared with original bacterium, this mutagenic strain is significantly less than starting strain in form.Starting strain yeast saccharomyces cerevisiae CICC31481 is purchased from Chinese industrial microbial strains preservation administrative center.Yeast strain of the present invention adopts following flow process to carry out seed selection: original bacterial classification → test tube activation → ethyl sulfate (DES) mutagenesis → height that sets out oozes plate screening → nitrosoguanidine (NTG) mutagenesis screening → height and oozes the test of sieve again → mitotic stability of dull and stereotyped primary dcreening operation → shaking flask test → 7L fermentor tank.CGMCCNo.4429 glucose-tolerant concentration can reach 300g/L.The present invention uses bacterial classification to see that name of patent application is preservation of bacteria strain in the application of a strain osmophilic yeast bacterium, number of patent application 201110105966, and open day is 2011.09.14.
Lactobacillus rhamnosus (Lactobacillus rhamnosus) CGMCC No.4430 bacterial strain feature is 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 oyster white, and smooth surface is moistening, thickness, and edge is more neat.Compared with original bacterium, this mutagenic strain is significantly less than starting strain in form.Starting strain lactobacillus rhamnosus CGMCC No.1.2134 is purchased from China Committee for Culture Collection of Microorganisms's common micro-organisms center.Lactobacillus rhamnosus of the present invention adopts following flow process to carry out seed selection: original sieve again → mitotic stability of bacterial classification → test tube activation → high temperature acclimation → ethyl sulfate (DES) mutagenesis → high sugared plate screening → nitrosoguanidine (NTG) mutagenesis screening → high temperature bacterium screening → shaking flask test → 5L fermentor tank test of setting out.Object bacterial strain CGMCC No.4430 is done to the experiment of 5L lactic acid fermentation tank, and result shows: compared with starting strain, CGMCC No.4430 glucose-tolerant concentration can reach 270g/L, has improved 95% compared with original bacterium; After fermentation ends, lactic acid content is 60g/L, has improved 158% compared with original bacterium.Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.4429, lactobacillus rhamnosus (Lactobacillus rhamnosus) CGMCC No.4430 depositary institution are China Committee for Culture Collection of Microorganisms's common micro-organisms centers, preservation date: on December 8th, 2010.Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101.
Aspergillus niger (Aspergillus niger) CGMCC No.3.5269, Bacillus licheniformis (Bacillus licheniformis) CGMCC No.1.813 buy in Chinese common micro-organisms culture presevation administrative center.
In the present invention, aspergillus niger spore adopts blood cell plate counting process, and other bacterial classification adopts microorganism plate count method to detect viable count.
What in the present invention, microbial inoculum was composite compared with ratio of greater inequality example is: aspergillus niger 25-40 part, Bacillus licheniformis 10-20 part, yeast saccharomyces cerevisiae 15-20 part, lactobacillus rhamnosus 25-35 part.
Invention product adopts the microbial inoculum compound proportion technology of scientific research effectively to guarantee reasonable constituents and the ratio of various microbiobacterial 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 feed, and the yield ratio of unit consumption feed has had and significantly improved than contrast, and the output of milk cow and quality are improved and ensure.
The stable performance of invention product, use safety, with other fodder additives all without incompatibility.Product of the present invention is stable and controllable for quality, non-environmental-pollution, and can improve immunizing power and can improve culture benefit.Product meets the requirement of green feed additive completely.The day weight gain that is significantly improved and feed conversion rate effect.
In product of the present invention, lactobacillus rhamnosus and yeast saccharomyces cerevisiae have booster action to the digestion of animal gastrointestinal tract, yeast saccharomyces cerevisiae for the growth and breeding of rumen microorganism provides useful amino acid, vitamin B group and other trace element, increases the resultant quantity of rumen microbial protein matter in cud.Lactobacillus rhamnosus discharges at the small intestine of milk cow, can consume the oxygen in animal intestinal, pathogenic bacterium harmful in enteron aisle are suppressed as aerobic bacteria (being mainly intestinal bacteria, Salmonellas etc.), can also suppress the synthetic of ammonia and amine, benefit strengthening immunity, reach and improve milk cow gi tract microbial ecological balance, be conducive to object healthy and raising production performance; In the enzyme that Bacillus licheniformis produces in enteron aisle metabolism and fermentation of Aspergillus niger culture cellulase, dextranase, lignin decomposition enzyme and the amylase of the effective dose that contains be conducive to the ruminant feeds such as milk cow in the digesting and assimilating of Fibrous feedstuff, improve the microecological balance of intestine of ruminants, improve efficiency of feed utilization, improve milk production of cow, improve the quality of milk, improve milk cow body immunity, reduce intestinal tract disease, purify feeding environment.This fodder additives can be applicable to milk cattle cultivating industry, can significantly improve milk crop and quality.
Product of the present invention can improve milk cow body immunity, reduces the sickness rate of latent mammitis, intestinal tract disease.At present, in China milk cows, latent mammitis sickness rate is higher, once milk cow suffers from mastitis just must injection of antibiotics treatment, 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 to harm).Use product of the present invention can reduce bovine subclinical mastitis sickness rate, feeding experiment shows that product of the present invention reduces mastitis sickness rate 70% (with control group comparison), obviously reduce the generation of mammitis of cow, reduce the antibiotic output that uses and contain microbiotic milk, indirectly improved culturist's benefit.
The level that product of the present invention is produced improving China milk cow, the aspect such as effective utilization (improve the utilization ratio of cheap roughage, improve the utilization ratio of nutritive substance in feed etc.) that fully improves feed resource all has very high value.
The present invention is also applicable to other ruminating animals feeding as beef cattle, sheep.
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, Bacillus licheniformis is 1.0～3.0 × 10
8individual/gram, yeast saccharomyces cerevisiae 1.0～3.5 × 10
8individual/gram, lactobacillus rhamnosus 1.3～4.5 × 10
8individual/gram.Microbial inoculum is composite: various microbial inoculum compound proportions, 30 parts of aspergillus nigers, 20 parts of Bacillus licheniformis, 20 parts of yeast saccharomyces cerevisiaes, 30 parts of lactobacillus rhamnosus, composite microbial inoculum packing.
Embodiment 2: high-efficiency activated feedstuff additive product
Product is composed as follows: aspergillus niger spore concentration 5 × 10
8individual/gram, Bacillus licheniformis is 5 × 10
8individual/gram, yeast saccharomyces cerevisiae 6 × 10
8individual/gram, lactobacillus rhamnosus 6 × 10
8individual/gram.
Microbial inoculum is composite: various microbial inoculum compound proportions, 40 parts of aspergillus nigers, 15 parts of Bacillus licheniformis, 16 parts of yeast saccharomyces cerevisiaes, 29 parts of lactobacillus rhamnosus, composite microbial inoculum packing.
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 microbiobacterial agent.
The production of microbiobacterial agent:
The preparation method of aspergillus niger spore
1) slant culture method
Slant medium 10mL adds test tube, and 121 ℃, after 20min sterilizing, pendulum inclined-plane, cooling, inoculation.30 ℃ of cultivations are paved with inclined-plane to black spore.
2) K formula culturing bottle spore
Get 10 ° of Brix worts and add 2% agar, pack 500mL K formula culturing bottle into, 121 ℃, after 20min sterilizing, paving inclined-plane is cooling.Access spore suspension 1mL, guarantees that suspension is inoculated in whole media surface; Be sidelong into thermostat container, 30 ℃ of cultivations are paved with inclined-plane to black spore.
3) solid-state amplification culture
K formula phialosporae is made to spore suspension, and spore concentration is greater than 1.0 × 10
8individual/mL.Get 200kg solid medium (wheat bran 140kg, 10 ° of Brix wort 60L), after fully mixing, put into tray, sterilizing 1 hour at 121 ℃.After cooling, access spore suspension, stirs.Culture temperature is controlled at 30 ℃, humidity 80-90%, every 10 hours stirrings once, incubation time 3 days; Treat that culture material covers with spore and can finish to cultivate
Drying and crushing: after fermentation ends, tray is placed on to fluidised bed drying, drying temperature is controlled at 60 ℃, will lower than below 10% time, pulverize solid culture medium with pulverizer until moisture content of material, and crushing material aperture is more than 100 orders.
The preparation method of Bacillus licheniformis
Substratum composition (g/L): glucose 60, yeast extract 20, dipotassium hydrogen phosphate 0.5, magnesium sulfate heptahydrate 0.5, pH6.8.
(1) first order seed is cultivated: 500mL shaking flask liquid amount is 100mL, accesses a ring Bacillus licheniformis after sterilizing, on shaking table, and 180 revs/min, 37 ℃ of culture temperature, incubation time 24 hours is to logarithmic phase;
(2) secondary seed is cultivated: first order seed is accessed according to the inoculum size of 10% (v/v) in the seeding tank of 30L, liquid amount 21L, 37 ℃ of culture temperature, 200 revs/min of stirring velocitys, ventilation (V/V) 1: 0.5, tank pressure 0.05Mpa, incubation time 24 hours is to logarithmic phase.
(3) fermentor cultivation: the seed in seeding tank is accessed to 300L fermentor tank with 10% (v/v) inoculum size, liquid amount 210L, 37 ℃ of culture temperature, 200 revs/min of stirring velocitys, ventilation (V/V) 1: 0.5, tank pressure 0.05Mpa, incubation time 24 hours, cultivates and finishes bacteria concentration 5.0 × 10
9individual/mL.
(4) centrifugation: adopt Plate Filtration to separate and obtain thalline.
(7) vacuum lyophilization: adopt drying process with atomizing to process acquisition dry bacteria after adding protective material; Protective material consists of skimmed milk 10%, lactose 5%, glycerine 5%.
The preparation method of S. cervisiae:
From inclined-plane switching S. cervisiae to liquid shaking bottle, liquid shaking bottle is transferred and is carried out enlarged culturing into fermentor tank after three grades spread cultivation, and cultivates complete cryoconcentration, mixes with carrier, and fluid bed drying process obtains active yeast saccharomyces cerevisiae microbial inoculum.
The cultivation of brewing yeast cell: adopt the slant strains acquisition brewing yeast cell that spreads cultivation step by step;
(1) first order seed is cultivated: by yeast saccharomyces cerevisiae slant strains access 500mL shaking flask, and substratum loading amount 100mL, 120 revs/min of rotary shaking tables, 30 ℃ of culture temperature, incubation time 18 hours;
(2) secondary seed is cultivated: by first order seed, according in 10% inoculum size access 500mL secondary seed shaking flask, culture condition is identical with first order seed;
(3) three grades of seed culture: secondary seed is accessed in tri-grades of seed shaking flasks of 5000mL to substratum loading amount 1000mL, 100 revs/min of rotary shaking tables, 30 ℃ of culture temperature, incubation time 18 hours with 10% inoculum size;
(4) first class seed pot is cultivated: the first class seed pot by three grades of seeds take 5% inoculum size access cubic capacity as 150L, fermention medium loading amount 100L, 30 ℃ of culture temperature, 100 revs/min of stirring velocitys, ventilation (V/V) 1: 0.5, tank pressure 0.05Mpa, incubation time 16 hours;
(5) fermentor cultivation: first class seed pot bacterial classification is accessed to cubic capacity as 3 tons of secondary seed tanks take 5% inoculum size (v/v), 2 tons of fermention medium loading amounts, 30 ℃ of culture condition culture temperature, 100 revs/min of stirring velocitys, ventilation in early stage (V/V) 1: 0.5, tank pressure 0.05Mpa, in earlier stage incubation time 12 hours; Later stage tank pressure 0.05Mpa, 50 revs/min of stirring velocitys, incubation time 8 hours, the yeast saccharomyces cerevisiae concentration of cultivating latter stage reaches 3.0 × 10
9individual/mL.
(6) concentrated: fermented liquid is concentrated in vacuo to 45% of original volume through low-temperature negative-pressure, obtains S. cervisiae concentrated solution.
(7) add carrier: in S. cervisiae concentrated solution, add the carrier mixing, mix; The weight ratio of concentrated solution and carrier is 0.5-0.7: 1, and vehicle group becomes: CaCO
340 parts, 20 parts, dextrin, 20 parts of Zein powders.
(8) dry: fluidised bed drying, 50 ℃ of drying temperatures.
Substratum adopts 10% malt extract medium or adopts the molasses culture medium of adding the inorganic salt such as ammonium sulfate, specifically cultivates production technique referring to Xiao Dongguang " production of Active Dry Yeast and utilisation technology ".
The preparation of lactobacillus rhamnosus microbial inoculum:
Slant strains access triangular flask, transfers in fermentor tank after being cultured to logarithmic phase, and controlling temperature is 45 ℃, and ventilating ratio is 1: 0.1 (v/v), cultivates about 18 hours to logarithmic phase.Ferment complete through Plate Filtration separate obtain wet thallus, add the protective material that consists of 10% skimmed milk, 5% trehalose, 5% glycerine, obtain microbial inoculum by lyophilize, moisture content is lower than 10%.
The experiment of product effect:
Selection and the test design of test ox: tested 7 days-October 7 September in 2010 and carry out in Wuzhong City kumquat cattle farm, Ningxia, random pair principle is taked in this test, by random 40 oxen test group and the control group of being divided into, every group of 20 oxen, in daily ration, add product 40g by every every day, feed 60 days, result shows experimental group day weight gain 972.7g, control group day weight gain 746.8g, difference is (P < 0.01) extremely significantly.Feed after 5 days, its ight soil dry-matter 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 test ox has spent peak of lactation), milk fat content has also increased, and effect is fine.From milk cow appearance, the hair of the milk cow of hello high-efficiency biological active fodder additives is bright, and appetite is good, and mastitis sickness rate reduces.
Claims (4)
1. a biological active fodder additives products, comprises yeast saccharomyces cerevisiae, it is characterized in that: in every gram of product, contain aspergillus niger spore 1.0～9.0 × 10
8individual, Bacillus licheniformis 1.0～9.0 × 10
8individual, yeast saccharomyces cerevisiae 1.0～8.5 × 10
8individual, lactobacillus rhamnosus 1.3～8.5 × 10
8individual, described aspergillus niger is that CGMCC No.3.5269, Bacillus licheniformis are that CGMCCNo.1.813, yeast saccharomyces cerevisiae are that CGMCC No.4429, lactobacillus rhamnosus are CGMCC No.4430.
2. biological active fodder additives products according to claim 1, is characterized in that: described aspergillus niger spore, Bacillus licheniformis, yeast saccharomyces cerevisiae, lactobacillus rhamnosus compound proportion are aspergillus niger 25-50 part, Bacillus licheniformis 10-25 part, yeast saccharomyces cerevisiae 15-25 part, lactobacillus rhamnosus 25-40 part.
3. biological active fodder additives products according to claim 1, is characterized in that every gram of product contains aspergillus niger spore 1.0～3.0 × 10
8individual, Bacillus licheniformis 1.0～3.0 × 10
8individual, yeast saccharomyces cerevisiae 1.0～3.5 × 10
8individual, lactobacillus rhamnosus 1.3～4.5 × 10
8individual.
4. biological active fodder additives products according to claim 1, is characterized in that every gram of product contains aspergillus niger spore 5 × 10
8individual, Bacillus licheniformis is 5 × 10
8individual, yeast saccharomyces cerevisiae 6 × 10
8individual, lactobacillus rhamnosus 6 × 10
8individual.
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