CN101112397A - Viable prebiotics bacteria microcapsule preparations for livestock and method for preparing the same - Google Patents
Viable prebiotics bacteria microcapsule preparations for livestock and method for preparing the same Download PDFInfo
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- CN101112397A CN101112397A CNA2007100586272A CN200710058627A CN101112397A CN 101112397 A CN101112397 A CN 101112397A CN A2007100586272 A CNA2007100586272 A CN A2007100586272A CN 200710058627 A CN200710058627 A CN 200710058627A CN 101112397 A CN101112397 A CN 101112397A
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Abstract
The present invention relates to a beneficial bacteria microcapsule preparation used for livestock and the preparation method. The invention is composed of core material, bacterial suspension, embedding material and carrier; wherein, the ratio of the parts by weight of the bacterial suspension and the core material is 1:2; the volume ratio of the mixed liquor which is formed by the core material and the bacterial suspension and the embedding material is 1:3. The bacterial suspension is prepared by the microbial fermentation and the centrifugation, the microcapsule is made by the absorption of the core material and embedding, then the living bacterial microcapsule is made by freezing and drying, and the living bacterial microcapsule preparation is prepared according to the different animals and different growth stages. The main technology is characterized by safe usage, no residue, no drug resistance, high survival rate in the small intestine of the animals and long survival time, which can be used as the feed additive or the veterinary drug to treat diarrhea of the animals.
Description
Technical field
The invention belongs to the microorganism formulation technical field, relate to a kind of prebiotics bacteria microcapsule preparations that is used for domestic animal and preparation method thereof, also relate to the preparation method of viable bacteria microcapsule formulation simultaneously.
Background technology
Nineteen sixty-five Lilley and Stillwell at first use probiotics (Probiotics), and it is defined as " by a kind of microorganism secretion, stimulating the material of another kind of growth of microorganism ".1974, Parker thinks " probiotics is a microbiological materials of keeping microbial balance in the intestinal ". probiotic bacteria has overcome the particularly negative effect brought of antibiotic of non-nutritive additive, it is pollution-free, noresidue, anti-disease, promotes the natural additive of growth of animal, has obtained using widely in feed industry.It is generally acknowledged that probiotic bacteria just is meant can survive in organism (mainly being humans and animals), and a quasi-microorganism useful to host's life and health generally is called probiotics to probiotics viable bacteria preparation or the microorganism formulation that contains culture of microorganism.First people just begin to recognize that microorganism and product thereof can exert an influence to body from eighties of last century, and screening has obtained many microorganisms that are considered to probiotic bacteria from microorganisms such as antibacterial, fungus, yeast.Probiotic bacteria is mainly used in the balance of keeping microecosystem in the animal body, does not promptly set up as yet under the situation of good intestinal microflora children domestic animal in age, uses probiotic bacteria to reduce the animal production loss; Probiotic bacteria can also partly substitute antibacterials, by helping to recover the balance of microecosystem in the animal body, reaches the purpose of treatment Animal diseases.Probiotic bacteria commonly used at present has lactobacillus, Bacillus coagulans etc., and its beneficial effect is:
1, to the Nutrition of animal: lactobacillus can synthesize the required multivitamin of animal body, as thiamine, riboflavin, nicotinic acid, pyridoxol, pantothenic acid, folic acid, vitamin B12 etc.Estimate that the microorganism on the caecum can provide 25%～30% of animal vitamin needs amount, can satisfy the needs of body to vitamin substantially.The volatile fatty acid that lactobacillus produces also makes intestinal pH value and redox potential reduce for big intestinal mucosa metabolism provides the energy, helps the absorption of mineral calcium, ferrum, magnesium, zinc etc.
2, suppress the breeding of pathogen: acid products such as the lactic acid that lactobacillus produces, acetic acid can suppress the pathogen growth and breeding.Lactobacillus can produce the plain material of bacterioid, and pathogen is had inhibitory action.The molecular weight that the bacillus acidophilus produces is 3500 peptide, can suppress the synthetic of e. coli dna, can suppress the growth of a series of Gram-positives and negative pathogen.The bacillus acidophilus can produce H
2O
2, the growth and breeding of pathogenic bacterium such as inhibition staphylococcus can also produce biacetyl, and this chemical compound has inhibitory action to many pathogen.Bacillus cereus can produce volatile fatty acids such as acetic acid, propanoic acid, butanoic acid in its growth course, these acids can reduce the animal intestinal pH value, can effectively suppress the pathogen growth.Produce a small amount of antibiotic substance one bacitracin, harmful microorganism is risen suppress and killing action.Bacillus cereus can consume a large amount of oxygen, keeps the intestinal anaerobic environment, suppresses the pathogenic bacterium growth, and the normal ecological balance of intestinal is kept in the effect of balance and stability lactobacillus.
3, improve animal immunizing power: with the lactobacillus fermentation product ablactational baby pig of feeding, the IgG level obviously raises in the piglet serum.
4, the characteristics of Bacillus coagulans and effect show: good stability.Acidproof, salt tolerant, high temperature resistant (100 ℃) and anti-extruding in pelletization and all can keep high stability by the acid stomach environment, can produce vitamin.Bacillus cereus can produce vitamin B group such as B1, B2, B6 and vitamin C, vitamin K in the growth and breeding process, for animal provides vitamin.Produce multiple digestive enzyme: Bacillus coagulans has the very strong protease of secretion, lipase and amylase activity, thereby can replenish the deficiency of intestinal endogenous enzymes.Can also produce phytase, promote animal the utilization of phytate phosphorus and digesting and assimilating to fat.The amino oxidase that produces and the enzyme of decomposing hydrogen sulfide can be oxidized to indoles nontoxic, harmless material, thereby reduce the concentration and the stink of ammonia, hydrogen sulfide in the poultry house, reduce environmental pollution.
About the research of thalline microcapsule, be the advanced technology that develops rapidly in recent years, it is widely used in many fields with own unique advantage.The micro encapsulation that just has the eighties in 20th century researcher to carry out lactobacillus is explored, Pranee carries out micro encapsulation as the wall material to lactobacillus with sodium alginate, the microcapsule of gained is used for the production of lactic acid, and fertility performance is good, and this lactobacillus micro-capsule can be reused.External at present relevant laboratory report can utilize lactobacillus micro-capsule cell mixing probiotics leaven, yoghourt, extracellular polysaccharide, lactic acid, the production of bacteriocin.
Summary of the invention
One object of the present invention is to provide by Bacillus coagulans, bacillus acidophilus, Lactobacillus plantarum, lactobacillus lactis, streptococcus faecalis and the carrier prebiotics bacteria microcapsule preparations by different proportioning combinations.
Another object of the present invention is, the preparation method of prebiotics bacteria microcapsule preparations is disclosed, adopt the method for biofermentation, prepare through microcapsule, the viable bacteria microcapsule is made in lyophilization then, and then, make the viable bacteria microcapsule formulation and survive bacteria microcapsule preparations according to different animals and different growth stage proportioning mix homogeneously.
Further object of the present invention has been to disclose the application of prebiotics bacteria microcapsule preparations aspect treatment animal alimentary canal diseases medicine.Comprise the application of treatment young stock diarrhoea, dysentery medicine aspect.Wherein said young stock mainly refers to piglet, chick, childhood milch cow or the like, also can be simultaneously grow up pig, become chicken, milch cow grows up.
A present invention also purpose has been to disclose the application of prebiotics bacteria microcapsule preparations aspect suffering from diarrhoea and build up one's resistance to disease as feed additive prevention young stock.
For achieving the above object the technical solution adopted in the present invention: a kind of prebiotics bacteria microcapsule preparations that is used for domestic animal is characterized in that it is made up of core, bacteria suspension, embedded material and carrier by each single bacterium; Wherein the ratio of weight and number of bacteria suspension and core is 1: 2; The volume ratio of formed mixed liquor of core and bacteria suspension and embedded material is 1: 3; The bacteria suspension of each single bacterium is: Bacillus coagulans, bacillus acidophilus, Lactobacillus plantarum, lactobacillus lactis and streptococcus faecalis; Viable bacteria content is respectively: bacillus acidophilus's microcapsule 〉=1.0 * 10
9Cfu/g; Lactobacillus plantarum microcapsule 〉=1.0 * 10
9Cfu/g; Bacillus coagulans microcapsule 〉=1.0 * 10
9Cfu/g; Lactobacillus lactis microcapsule 〉=1.0 * 10
9Cfu/g; Streptococcus faecalis microcapsule 〉=1.0 * 10
9Cfu/g.
Viable bacteria microcapsule formulation of the present invention, wherein said core are 1: 1 micropore starch and lactose (ratio of weight and number) mixture; Embedded material is the sodium alginate soln of 3-4%; The concentration of calcium chloride is the 3-4% aqueous solution; Carrier is 1-1.5 part zein.
Viable bacteria microcapsule formulation of the present invention, the viable bacteria total content that it is characterized in that described active bacteria formulation is 1.0 * 10
9More than the cfu/g, microcapsule diameter is 1～2mm.
The preparation method of viable bacteria microcapsule formulation of the present invention, carry out as follows:
(1) slant strains is cultivated: each strain uses corresponding culture medium and adds 1.5-2.5% agar, and static cultivation is 20-48 hour under 30-40 ℃ of condition, puts into 4 ℃ of refrigerators and preserves;
(2) the liquid first order seed is cultivated: be shake-flask culture, get the 250ml triangular flask and add seed culture medium 50-100ml, get inclined-plane seed strain 1 ring, insert in the culture medium 30-40 ℃, 100-220r/minpm cultivates 30h;
(3) the liquid secondary is cultivated: 10L automated seed fermentation tank, and dress seed culture medium 5-8L meets weight 5-10%, 30-40 ℃, mixing speed 50-100 rev/min, cultivates 30h;
(4) each fermentation tank single strain pure culture condition: temperature 25-40 ℃, ventilation 9-13m
3/ h, tank pressure 0.02-0.09MPa, inoculum concentration 1.5-7.5%, time 19-72 hour, the fermentation liquid viable count reached 10
8-10
9Cfu/ml;
The fermentation medium of each strain (g/L):
The bacillus acidophilus: tryptone 2.5-7.5g, peptone 5-15g, glucose 5-15g,, lactose 2.5-7.5g, yeast extract powder 4-10g pH7.0;
Streptococcus faecalis: peptone 10-30g, Carnis Bovis seu Bubali cream 10-30g, lactose 10-30g, pH6.8 ± 0.2;
Bacillus coagulans: peptone 5-15g, yeast extract 5-15g, glucose 3-9g, MgSO
47H
2O 0.5-1.5g, K
2HPO
41-3g, MnSO
450 μ g, pH7.0;
Lactobacillus plantarum: peptone 5-15g, Carnis Bovis seu Bubali cream 5-15g, yeast extract 3-7g, glucose 15-25g, disodium citrate 1.5-3.5g, K
2HPO
41-3g, sodium acetate 2.5-7.5g, MgSO47H
2O1-3g, MnSO
44H
2O 0.01g tween 10ml, pH6.0-6.5;
Lactobacillus lactis: tryptone 3.5-6.5g, peptone 7.5-12.5g, glucose 7.5-12.5g,, lactose 2.5-7.5g, yeast extract powder 5-9g, pH7.0;
(5) preparation of viable bacteria microcapsule: adopt above-mentioned each thalline of the centrifugal collection of continuous high speed centrifuge, add sterilized water and be mixed with 1.0 * 10
11The cfu/mL bacteria suspension, add then micropore starch and lactose (1: 1W/W) mixture, ratio are 1: 2V/W, evenly stir 100-200r/min 30-60min; Adding 3-4% sodium alginate sterile solution ratio at last is 1: 3V/V, by the capsule automatic generator microgranule is splashed in the aseptic calcium chloride solution of 3-4%, and solidify 1-2h, make the viable bacteria microcapsule;
(6) filter collection viable bacteria microcapsule ,-50 ℃ of pre-freeze 2～3h, vacuum lyophilization makes exsiccant viable bacteria microcapsule;
(7) count plate: measuring exsiccant viable bacteria microcapsule viable bacteria content is 1.0 * 10
9More than the cfu/g, water content is lower than 8%; Microcapsule diameter is 1～2mm.
(8) the viable bacteria microcapsule that makes is added 1-1.5 part carrier zein, by different domestic animal demand compatibility proportioning mix homogeneously, make the viable bacteria microcapsule formulation then, as feed additive, or as treatment diarrheal veterinary drug.Viable bacteria microcapsule wherein is made up of the prebiotics bacteria microcapsule of following portions by weight: Bacillus coagulans microcapsule 0.5-1 part; 1～1.8 part of bacillus acidophilus's microcapsule; 1～1.5 part of Lactobacillus plantarum microcapsule; Lactobacillus lactis microcapsule 0.5-1.5 part; Streptococcus faecalis microcapsule 0.5-1 part; Wherein the viable bacteria content of each component is respectively: bacillus acidophilus's microcapsule 〉=1.0 * 10
9Cfu/g; Lactobacillus plantarum microcapsule 〉=1.0 * 10
9Cfu/g; Bacillus coagulans microcapsule 〉=1.0 * 10
9Cfu/g; Lactobacillus lactis microcapsule 〉=1.0 * 10
9Cfu/g; Streptococcus faecalis microcapsule 〉=1.0 * 10
9Cfu/g.
Existing probiotics viable bacteria preparation, in general thalline does not all pass through microcapsule embeddedly, just adds protective agent; be lyophilized into powder, directly be packaged as feed additive, or pack with general hard-shell capsule; directly do not pack, be made into large capsule through the mycopowder of micro encapsulation.Here used general capsule wall material makes probiotic bacteria can not discharge rapidly in intestinal, grows surely, is unfavorable for suppressing the growth of harmful bacterium, thus the balance that can not regulate the body intestinal microbial population effectively; Perhaps, in stomach, discharge, because thalline is without micro encapsulation, so very fast by the acid in the gastric juice, digestive enzyme, antibiotic deactivation, can not successfully arrive intestinal, and the probiotic bacteria of micro encapsulation can effectively solve gastric acid, and digestive enzyme and antibiotic etc. are to the problem of the microbial deactivation of activity, makes probiotic bacteria successfully arrive intestinal and discharges immediately and grow surely.
The present invention further discloses the application of prebiotics bacteria microcapsule preparations aspect suffering from diarrhoea and build up one's resistance to disease as feed additive prevention young stock.
Be prebiotics bacteria microcapsule preparations proportioning (table 1) below at different animals
| Animal | Bacillus acidophilus's microcapsule (part) | Streptococcus faecalis microcapsule (part) | Lactobacillus plantarum microcapsule (part) | Bacillus coagulans microcapsule (part) | Lactobacillus lactis microcapsule (part) | Amount to (gram) |
| Piglet | 1.5 | 1 | 1 | 1 | 1.5 | 1 |
| Pig grows up | 1 | 0.5 | 1 | 0.5 | 1 | 1 |
| Chick | 1.8 | 1.5 | 1 | 1 | 1 | 1 |
| Become chicken | 1.5 | 1 | 1 | 1.5 | 1 | 1 |
| Calf | 1 | 1 | 1.5 | 0.5 | 0.5 | 1 |
| Milch cow grows up | 1.5 | 0.5 | 1 | 0.5 | 1 | 1 |
Experimental result:
As the feed additive prevention and the usage of building up one's resistance to disease: prebiotics bacteria microcapsule preparations joins in the chick daily ration with the 0.1-0.2% of daily ration weight, and chick goes out behind the shell to begin to feed in the second-three day and stopped to the 15th day.Prebiotics bacteria microcapsule preparations joins in the piglet diet with the 0.02-0.04% of daily ration weight, and piglet birth back began to feed on the 5th day, fed continuously and took 20 to 30 days.0.03-0.06% with powdery concentrated feed weight in the order grain joins in the calf daily ration with prebiotics bacteria microcapsule preparations, from back the 7th day of calf birth, takes continuously 30 to 40 days.Significantly improve young stock body constitution, prevalences such as symptom of diarrhea and Hakuri are starkly lower than matched group.(table 2)
| Project | The young stock kind | A test number (number of elements) | Test period (my god) | Diarrhea disease percentage % | Average daily gain (gram) | Raising rate % on average increasing day by day | Feedstuff-meat ratio | Feedstuff-meat ratio reduction rate % |
| Matched group | Chick | 100 | 15 | 35 | 38.72 | 0 | 2.34 | 0 |
| Experimental group | Chick | 100 | 15 | 12 | 44.65 | 15.32 | 1.86 | 20.51 |
| Matched group | Piglet | 30 | 25 | 26.67 | 487.63 | 0 | 2.26 | 0 |
| Experimental group | Piglet | 30 | 25 | 6.67 | 523.79 | 7.42 | 1.93 | 14.60 |
| Matched group | Calf | 20 | 30 | 25 | 987.36 | 0 | 2.46 | 0 |
| Experimental group | Calf | 20 | 30 | 10 | 1036.85 | 5.01 | 2.12 | 13.82 |
The present invention further discloses the application of prebiotics bacteria microcapsule preparations aspect treatment animal alimentary canal diseases medicine.Comprise the application of treatment young stock diarrhoea, dysentery medicine aspect.
The diarrhea treatment consumption: after finding Huang, Hakuri and symptom of diarrhea, chick is dissolved in water by 1000 milligrams of prebiotics bacteria microcapsule preparations of per kilogram of body weight and gavages, and one day twice, eliminate up to disease, once-a-day continue to take two to three days then again.Become the chicken employing to be dissolved in water by per kilogram of body weight 400-500 milligram prebiotics bacteria microcapsule preparations and gavage, 1-2 light takes twice, takes later every day once continuing to take two to three days after disease is eliminated.Piglet is dissolved in water by per kilogram of body weight 100-200 milligram prebiotics bacteria microcapsule preparations and gavages, and one day twice, eliminate up to disease, once-a-day continue to take two to three days then again.Become pig to admix in a small amount of feedstuff, directly search for food by per kilogram of body weight 40-50 milligram prebiotics bacteria microcapsule preparations, one day twice, eliminate up to disease, once-a-day continue to take two to three days then again.Calf is admixed in a small amount of feedstuff by 500 milligrams of prebiotics bacteria microcapsule preparations of per kilogram of body weight, directly searches for food, and one day twice, eliminate up to disease, once-a-day continue to take two to three days then again.Being dissolved in water gavages, and one day twice, eliminate up to disease, once-a-day continue to take two to three days then again.Become cattle to admix in a small amount of feedstuff, directly search for food by 200 milligrams of prebiotics bacteria microcapsule preparations of per kilogram of body weight, one day twice, eliminate up to disease, once-a-day continue to take two to three days then again.
The present invention compares with present existing various prebiotics bacteria preparations, and the distinguishing feature and the good effect that are had are:
(1) the prepared prebiotics bacteria microcapsule preparations of the present invention, its reasonable recipe, the manufacture method science, the scope of application is extensive, and is simple to operate, grasps easily, cost is low, can be used for various poultry as the utilization rate of feed additive, also can be used as veterinary drug simultaneously and use, be mainly used in intestinal tract diseases such as treating young stock diarrhoea, dysentery and have the disease-resistant effect of the poultry of enhancing with the raising feed additive.
(2) micro encapsulation helps probiotic bacteria body thalli growth and metabolite production, and the thalline of micro encapsulation, plasmid keep stability to strengthen, and thalline opposing antibiotic, acidproof, the ability of bile tolerance all increases, make prebiotics bacteria pass through gastric shield, grow in intestinal surely.
(3) can avoid by trace element in the feedstuff through the probiotic bacteria of micro encapsulation, a series of oxidoreductions that metal ion, vitamin etc. cause, acid-base reaction are to the inhibitory action of activity of probiotic.
Figure of description:
Fig. 1 is a prebiotics bacteria microcapsule preparations processing technology flow chart.
The specific embodiment
In order to explain the present invention more fully, provide the preparation embodiment of following prebiotics bacteria preparation and preparation method thereof.These embodiments only are to explain rather than limit the scope of the invention.
Embodiment 1
The slant medium of the present invention used strain source and the corresponding use of strain sees the following form: (table 3)
| Sequence number | Bacterium name (Chinese, Latin) | Numbering | Slant medium and shake-flask culture base (g/L) |
| 1 | Bacillus coagulans Bacillus coagulans | IFFI10067 | Peptone 10g, Carnis Bovis seu Bubali cream 5g, sodium chloride 5g, pH7.2-7.4 |
| 2 | Bacillus acidophilus Lactobacillus acidophilus | IFFI06005 | Defatted milk powder 100g, pH6.0 |
| 3 | Lactobacillus plantarum Lactobacillus plantarum | IFFI06009 | Peptone 10g, sucrose 20g, yeast extract 10g, ammonium phosphate 5g, pH7.2-7.4 |
| 4 | Lactobacillus lactis Lactobacillus lactis | IFFI06064 | Defatted milk powder 100g, pH6.0 |
| 5 | Streptococcus faecalis Streptococcus faecalis | AS1.997 | Peptone 10g, sucrose 20g, yeast extract 10g, ammonium phosphate 5g, pH7.2-7.4 |
One, the preparation of Bacillus coagulans
(1) get the Bacillus coagulans kind in adding 1.5% agar, in the above-mentioned slant medium of inoculum concentration 5%, static cultivation is 20 hours under 25 ℃ of conditions, puts into 4 ℃ of refrigerators and preserves.(2) change over to and shake in the bottle, 25 ℃ of temperature, 80 rev/mins of rotating speeds, inoculum concentration 1% is cultivated 19h.(3) liquid secondary culture condition: change in the seed fermentation jar, dress seed culture medium 5L connects 5%, 30 ℃ of weight, and 10 rev/mins of mixing speeds are cultivated 30h.(4) fermentation tank single strain pure culture condition: Bacillus coagulans fermentation medium (g/L): peptone 5g, yeast extract 5g, glucose 3g, MgSO
47H
2O 0.5g, K
2HPO
41g, MnSO
450 μ g, pH7.0.25 ℃ of temperature, ventilation 9m
3/ h, tank pressure 0.02MPa, inoculum concentration 1.5%, 72 hours time, the fermentation liquid viable count reaches 10
8-10
9Cfu/ml; The centrifugal collection thalline of fermentation liquid.
Two, bacillus acidophilus's preparation:
(1) get the bacillus acidophilus in adding 1.5% agar, in the above-mentioned slant medium of inoculum concentration 5%, static cultivation is 20 hours under 25 ℃ of conditions, puts into 4 ℃ of refrigerators and preserves.(2) change over to and shake in the bottle, 25 ℃ of temperature, 180 rev/mins of rotating speeds, inoculum concentration 1 is cultivated 19h.(3) liquid secondary culture condition: change in the seed fermentation jar, dress seed culture medium 5L connects 5%, 30 ℃ of weight, and 10 rev/mins of mixing speeds are cultivated 30h.(4) fermentation tank single strain pure culture condition: bacillus acidophilus's fermentation medium (g/L): tryptone 2.5g, peptone 5g, glucose 5g,, lactose 2.5g, yeast extract powder 4g, pH7.0.25 ℃ of temperature, ventilation 9m
3/ h, tank pressure 0.02MPa, inoculum concentration 1.5%, 72 hours time, the fermentation liquid viable count reaches 10
8-10
9Cfu/ml; The centrifugal collection thalline of fermentation liquid.
Three, the preparation of Lactobacillus plantarum:
(1) get Lactobacillus plantarum in adding 1.5% agar, in the above-mentioned slant medium of inoculum concentration 5%, static cultivation is 48 hours under 35 ℃ of conditions, puts into 4 ℃ of refrigerators and preserves.(2) change over to and shake in the bottle, 25 ℃ of temperature, 180 rev/mins of rotating speeds, inoculum concentration 3% is cultivated 19h.(3) liquid secondary culture condition: change in the seed fermentation jar, dress seed culture medium 5L connects 5%, 30 ℃ of weight, and 10 rev/mins of mixing speeds are cultivated 30h.(4) fermentation tank single strain pure culture condition: Lactobacillus plantarum fermentation medium (g/L): peptone 5g, Carnis Bovis seu Bubali cream 5g, yeast extract 3g, glucose 15g, disodium citrate 1.5g, K
2HPO
41g, sodium acetate 2.5g, MgSO
47H
2O1g, MnSO
44H
2O0.01g tween 10ml, pH6.0.25 ℃ of temperature, ventilation 9m
3/ h, tank pressure 0.02MPa, inoculum concentration 1.5%, 72 hours time, the fermentation liquid viable count reaches 10
8-10
9Cfu/ml; The centrifugal collection thalline of fermentation liquid.
Four, the preparation of lactobacillus lactis:
(1) the extracting lactic acid lactobacillus is in adding 1.5% agar, and in the above-mentioned slant medium of inoculum concentration 5%, static cultivation is 48 hours under 35 ℃ of conditions, puts into 4 ℃ of refrigerators and preserves.(2) change over to and shake in the bottle, 25 ℃ of temperature, 180 rev/mins of rotating speeds, inoculum concentration 3% is cultivated 19h.(3) liquid secondary culture condition: change in the seed fermentation jar, dress seed culture medium 5L connects 5%, 30 ℃ of weight, and 10 rev/mins of mixing speeds are cultivated 30h.(4) fermentation tank single strain pure culture condition: lactobacillus lactis fermentation medium (g/L): tryptone 3.5g, peptone 7.5g, glucose 7.5g, lactose 2.5g, yeast extract powder 5g, pH7.0.25 ℃ of temperature, ventilation 9m
3/ h, tank pressure 0.02MPa, inoculum concentration 1.5%, 72 hours time, the fermentation liquid viable count reaches 10
8-10
9Cfu/ml; The centrifugal collection thalline of fermentation liquid.
Five, the preparation of streptococcus faecalis:
(1) get streptococcusfaecium enterococci strain in adding 2.5% agar, in the above-mentioned slant medium of inoculum concentration 10%, static cultivation is 20 hours under 35 ℃ of conditions, puts into 4 ℃ of refrigerators and preserves.(2) change over to and shake in the bottle, 25 ℃ of temperature, 80 rev/mins of rotating speeds, inoculum concentration 3% is cultivated 19h.(3) liquid secondary culture condition: change in the seed fermentation jar, dress seed culture medium 5L connects 5%, 30 ℃ of weight, and 10 rev/mins of mixing speeds are cultivated 30h.(4) fermentation tank single strain pure culture condition: streptococcus faecalis fermentation medium: peptone 10g, Carnis Bovis seu Bubali cream 10g, lactose 10g, pH6.8 ± 0.2.25 ℃ of temperature, ventilation 9m
3/ h, tank pressure 0.02MPa, inoculum concentration 1.5%, 72 hours time, the fermentation liquid viable count reaches 10
8-10
9Cfu/ml; The centrifugal collection thalline of fermentation liquid.
Embodiment 2
Slant strains cultivation → liquid first order seed among the embodiment (shaking bottle) cultivation → liquid secondary is cultivated the 1 identical no longer repetition with embodiment.
(1) preparation of Bacillus coagulans:
Fermentation tank single strain pure culture condition: Bacillus coagulans fermentation medium (g/L): peptone 15g, yeast extract 15g, glucose 9g, MgSO
47H
2O 1.5g, K
2HPO
43g, MnSO
450 μ g, pH7.0.40 ℃ of temperature, ventilation 13m
3/ h, tank pressure 0.09MPa, inoculum concentration 7.5%, 19 hours time, the fermentation liquid viable count reaches 10
8-10
9Cfu/ml; The centrifugal collection thalline of fermentation liquid.
(2) bacillus acidophilus's preparation:
Fermentation tank single strain pure culture condition: bacillus acidophilus's fermentation medium (g/L): tryptone 7.5g, peptone 15g, glucose 15g, lactose 7.5g, yeast extract powder 10g, pH7.0.40 ℃ of temperature, ventilation 13m
3/ h, tank pressure 0.09MPa, inoculum concentration 7.5%, 19 hours time, the fermentation liquid viable count reaches 10
8-10
9Cfu/ml; The centrifugal collection thalline of fermentation liquid.
(3) preparation of Lactobacillus plantarum:
Fermentation tank single strain pure culture condition: Lactobacillus plantarum fermentation medium (g/L): peptone 15g, Carnis Bovis seu Bubali cream 15g, yeast extract 7g, glucose 25g, disodium citrate 3.5g, K
2HPO
43g, sodium acetate 7.5g, MgSO
47H
2O 3g, MnSO
44H
2O 0.01g tween 10ml, pH6.5.40 ℃ of temperature, ventilation 13m
3/ h, tank pressure 0.09MPa, inoculum concentration 7.5%, 19 hours time, the fermentation liquid viable count reaches 10
8-10
9Cfu/ml; The centrifugal collection thalline of fermentation liquid.
(4) preparation of lactobacillus lactis:
Fermentation tank single strain pure culture condition: lactobacillus lactis fermentation medium (g/L): tryptone 6.5g, peptone 12.5g, glucose 12.5g,, lactose 7.5g, yeast extract powder 9g, pH7.0.40 ℃ of temperature, ventilation 13m
3/ h, tank pressure 0.09MPa, inoculum concentration 7.5%, 19 hours time, the fermentation liquid viable count reaches 10
8-10
9Cfu/ml; The centrifugal collection thalline of fermentation liquid.
(5) preparation of streptococcus faecalis:
Streptococcus faecalis fermentation medium: peptone 30g, Carnis Bovis seu Bubali cream 30g, lactose 30g, pH6.8 ± 0.2.40 ℃ of temperature, ventilation 13m
3/ h, tank pressure 0.09MPa, inoculum concentration 7.5%, 19 hours time, the fermentation liquid viable count reaches 10
8-10
9Cfu/ml; The centrifugal collection thalline of fermentation liquid.
Embodiment 3
The preparation of viable bacteria microcapsule:
(1) with the foregoing description 1 resulting each fermentation liquid, adopt above-mentioned each thalline of the centrifugal collection of continuous high speed centrifuge, add sterilized water and be mixed with 1.0 * 1011cfu/mL bacteria suspension, add micropore starch and lactose (1: 1W/W) mixture then, ratio is 1: 2V/W, evenly stir 100r/min, 30min; Add 3% sodium alginate sterile solution at last, ratio is 1: 2V/V, by the capsule automatic generator microgranule is splashed in the 3% aseptic calcium chloride solution, and leave standstill 1h, make the viable bacteria microcapsule;
(2) vacuum lyophilization: filter and collect the viable bacteria microcapsule ,-50 ℃ of pre-freeze 2h, vacuum lyophilization makes exsiccant viable bacteria microcapsule;
(3) count plate: count plate: measuring exsiccant viable bacteria microcapsule viable bacteria content is more than 1.0 * 109cfu/g, adds 1.5 parts of zein carriers and mixes;
(4) with the viable bacteria microcapsule that makes by different domestic animal demand proportioning mix homogeneously, make the viable bacteria microcapsule formulation, microcapsule diameter is 1mm.
Embodiment 4
With the foregoing description 1 resulting each fermentation liquid, adopt above-mentioned each thalline of the centrifugal collection of continuous high speed centrifuge, add sterilized water and be mixed with 1.0 * 10
11The cfu/mL bacteria suspension, add then micropore starch and lactose (1: 1W/W) mixture, ratio are 1: 2V/W, evenly stir 200r/min, 60min; Add 4% sodium alginate sterile solution at last, ratio is 1: 2V/V, by the capsule automatic generator microgranule is splashed in the 4% aseptic calcium chloride solution, and leave standstill 2h, make the viable bacteria microcapsule; Vacuum lyophilization: filter and collect the viable bacteria microcapsule ,-50 ℃ of pre-freeze 3h, vacuum lyophilization makes exsiccant viable bacteria microcapsule; Measuring exsiccant viable bacteria microcapsule viable bacteria content is 1.0 * 10
9More than the cfu/g, add 1 part of zein carrier and mix; By different domestic animal demands, the hard-shell capsule of packing into is made the viable bacteria microcapsule formulation with the viable bacteria microcapsule that makes, and microcapsule diameter is 1mm.
Embodiment 5
With the foregoing description 2 resulting each ultrafiltration and concentration liquid, adopt above-mentioned each thalline of the centrifugal collection of continuous high speed centrifuge, add sterilized water and be mixed with 1.0 * 10
11The cfu/mL bacteria suspension, add then micropore starch and lactose (1: 1W/W) mixture, ratio are 1: 2V/W, evenly stir 200r/min, 60min; Add 4% sodium alginate sterile solution at last, ratio is 1: 2V/V, by the capsule automatic generator microgranule is splashed in the 4% aseptic calcium chloride solution, and leave standstill 2h, make the viable bacteria microcapsule; Vacuum lyophilization: filter and collect the viable bacteria microcapsule ,-50 ℃ of pre-freeze 3h, vacuum lyophilization makes exsiccant viable bacteria microcapsule; Measuring exsiccant viable bacteria microcapsule viable bacteria content is 1.0 * 10
9More than the cfu/g, add 1.5 parts of zein carriers and mix; By different domestic animal demands, the proportioning mix homogeneously is made the viable bacteria microcapsule formulation with the viable bacteria microcapsule that makes, and microcapsule diameter is 2mm.
Embodiment 6
Get 1.5 parts of bacillus acidophilus's microcapsules by weight, 1 part of streptococcus faecalis microcapsule, 1 part of Lactobacillus plantarum microcapsule, 1 part of Bacillus coagulans microcapsule, 1.5 parts of lactobacillus lactis microcapsules, above-mentioned each component and 1.5 parts of zein carrier mix homogeneously are made prebiotics bacteria microcapsule preparations, are used for the prevention and the treatment of piglet diarrhea.
Embodiment 7
Get 1 part of bacillus acidophilus's microcapsule by weight, 1 part of streptococcus faecalis microcapsule.1.5 parts of Lactobacillus plantarum microcapsules, 0.5 part of Bacillus coagulans microcapsule, 0.5 part of lactobacillus lactis microcapsule, above-mentioned each component and 1 part of zein carrier mix homogeneously are made prebiotics bacteria microcapsule preparations, are used for prevention of calf diarrheal and treatment.
Embodiment 8
Contrast experiment's situation:
0.2% of viable bacteria microcapsule formulation chick daily ration weight joined in the daily ration stir, chick is the 2-3 age in days, allows the chick free choice feeding observe growing state.
Zoopery: divide A with 200 broiler, two groups of B, A group is matched group, the B group is experimental group, every group each 100, carries out the contrast experiment of growth of meat chicken speed.The B group is admixed the viable bacteria microcapsule formulation by weight 0.2% ratio in feedstuff, wherein the weight ratio of viable bacteria microcapsule and feedstuff is 2: 1000, makes viable count 〉=2 * 10 in the grain of whenever setting a date
6Individual.Experimental period is the 2-17 age in days.Result of the test is that the average daily gain of B group is higher than A group 15.32%, P＜0.01.
Embodiment 9
The survival condition of viable bacteria in the viable bacteria microcapsule:
The viable bacteria microcapsule formulation that embodiment 4-7 obtains was stored 60 days at normal temperatures, measured the prebiotics bacteria survival condition of microcapsule, the result is as follows: (table 4)
| Period of storage | 0 day | 60 days | 120 days | 180 days | 240 | 300 |
| Embodiment 4-5 | 100 | 95 | 88 | 86 | 84 | 82 |
| Embodiment 6-7 | 100 | 96 | 86 | 84 | 82 | 80 |
As can be known from the results, viable bacteria microcapsule formulation provided by the invention is very stable at normal temperatures, stores still to keep the prebiotics bacteria activity greater than 80% after 300 days.
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.
Claims (8)
1. prebiotics bacteria microcapsule preparations that is used for domestic animal is characterized in that it is made up of bacteria suspension, embedded material and the carrier of core, each single bacterium; Wherein the ratio of weight and number of bacteria suspension and core is 1: 2; The volume ratio of formed mixed liquor of core and bacteria suspension and embedded material is 1: 3; The bacteria suspension of each single bacterium is: Bacillus coagulans, bacillus acidophilus, Lactobacillus plantarum, lactobacillus lactis and streptococcus faecalis; Viable bacteria content is respectively: bacillus acidophilus's microcapsule 〉=1.0 * 10
9Cfu/g; Lactobacillus plantarum microcapsule 〉=1.0 * 10
9Cfu/g; Bacillus coagulans microcapsule 〉=1.0 * 10
9Cfu/g; Lactobacillus lactis microcapsule 〉=1.0 * 10
9Cfu/g; Streptococcus faecalis microcapsule 〉=1.0 * 10
9Cfu/g.
2. viable bacteria microcapsule formulation as claimed in claim 1, wherein said core are that ratio of weight and number is 1: 1 micropore starch and a milk-sugar mixture; Embedded material is the sodium alginate soln of 3-4%; Carrier is 1-1.5 part zein.
3. viable bacteria microcapsule formulation as claimed in claim 1 is characterized in that the viable bacteria total content is 1.0 * 10 in the described active bacteria formulation
9More than the cfu/g; Microcapsule diameter is 1～2mm.
4. the application of the described viable bacteria microcapsule formulation of claim 1 aspect suffering from diarrhoea and build up one's resistance to disease as feed additive prevention young stock.
5. the application of the described viable bacteria microcapsule formulation of claim 1 aspect preparation treatment young stock diarrhoea.
6. method for preparing each described viable bacteria microcapsule formulation of claim 1-5 is characterized in that carrying out as follows:
(1) cultivation of prebiotics bacteria: each thalline is used the respective ramp spawn culture, cultivate through the liquid first order seed, the liquid secondary is cultivated, the pure enrichment culture of fermentation tank single strain;
(2) preparation of viable bacteria microcapsule: adopt above-mentioned each thalline of the centrifugal collection of continuous high speed centrifuge, add sterilized water and be mixed with 1.0 * 10
11The cfu/mL bacteria suspension, add then micropore starch and lactose (1: 1W/W) mixture, ratio are 1: 2V/W, evenly stir 100-200r/min 30-60min; Adding 3-4% sodium alginate sterile solution ratio at last is 1: 3V/V, by the capsule automatic generator microgranule is splashed in the aseptic calcium chloride water of 3-4%, and solidify 1-2h, make the viable bacteria microcapsule;
(3) filter collection viable bacteria microcapsule ,-50 ℃ of pre-freeze 2～3h, vacuum lyophilization makes exsiccant viable bacteria microcapsule;
(4) count plate: measuring exsiccant viable bacteria microcapsule viable bacteria content is 1.0 * 10
9More than the cfu/g, add 1-1.5 part zein carrier and mix;
(5) with the viable bacteria microcapsule that makes by different domestic animal demands, the proportioning mix homogeneously is made the viable bacteria microcapsule formulation.
7. preparation method as claimed in claim 6, viable bacteria microcapsule wherein is made up of the prebiotics bacteria microcapsule of following portions by weight: Bacillus coagulans microcapsule 0.5-1 part; 1～1.8 part of bacillus acidophilus's microcapsule; 1～1.5 part of Lactobacillus plantarum microcapsule; Lactobacillus lactis microcapsule 0.5-1.5 part; Streptococcus faecalis microcapsule 0.5-1 part; Wherein the viable bacteria content of each component is respectively: bacillus acidophilus's microcapsule 〉=1.0 * 10
9Cfu/g; Lactobacillus plantarum microcapsule 〉=1.0 * 10
9Cfu/g; Bacillus coagulans microcapsule 〉=1.0 * 10
9Cfu/g; Lactobacillus lactis microcapsule 〉=1.0 * 10
9Cfu/g; Streptococcus faecalis microcapsule 〉=1.0 * 10
9Cfu/g.
8. preparation method as claimed in claim 6, wherein each fermentation tank single strain pure culture condition: temperature 25-40 ℃, ventilation 9-13m
3/ h, tank pressure 0.02-0.09MPa, inoculum concentration 1.5-7.5%, time 19-72 hour, the fermentation liquid viable count reached 10
8-10
9Cfu/ml;
The fermentation medium of prebiotics bacteria (g/L) wherein:
Bacillus coagulans: peptone 5-15g, yeast extract 5-15g, glucose 3-9g, MgSO
47H
2O0.5-1.5g, K
2HPO
41-3g, MnSO
450 μ g, pH7.0;
Lactobacillus plantarum: peptone 5-15g, Carnis Bovis seu Bubali cream 5-15g, yeast extract 3-7g, glucose 15-25g, disodium citrate 1.5-3.5g, K
2HPO
41-3g, sodium acetate 2.5-7.5g, MgSO47H
2O1-3g, MnSO
44H
2O 0.01g tween 10ml, pH6.0-6.5.
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