CN105002155A - Probiotics preparation for pigs and preparation method thereof - Google Patents
Probiotics preparation for pigs and preparation method thereof Download PDFInfo
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Abstract
The invention provides a novel probiotics preparation for pigs and a preparation method thereof. According to the probiotics preparation for the pigs, the feed utilization efficiency of animals can be improved, the cost is saved, the steady state of the internal environment of the alimentary canal in an animal body is greatly improved, the absorption and utilization of nutrients and the growth of the animals are promoted, the production performance, the immune performance and the reproductive performance of the animals are significantly improved, the production cost is lowered, the economical benefit is improved, and the good application prospect is achieved.
Description
Technical field
The present invention relates to probiotics and preparation method thereof, specifically, relate to boar probiotic agent and preparation method thereof.
Background technology
Antibiotic use to preventing disease, growth promotion, increasing economic efficiency plays vital role, simultaneously its side effect produced such as the residual etc. of the destruction of the appearance of superbacteria, autogenous infection and superinfection, intestinal microecology balance and animal products and environment brings serious threat to the health of livestock industry and the mankind.Therefore, research and development Substitutes For Antibiotic, for green, safety, efficiently fodder additives provide reliable technical guarantee.Probiotic bacterium is as a kind of biologically active additives of new type natural, there is the effect of microbiotic and enzyme, nontoxic, without resistance, noresidue, and have improve intestinal microflora balance, suppress the growth and breeding of spoilage organism and pathogenic bacteria, strengthening immunity, growth promoting effects, promote that animal absorbs and improves efficiency of feed utilization, alternative microbiotic is applied in breeding production.Faecalis mainly concentrates on enterococcus faecalis and faecium as the application of probiotic bacterium, and faecium belongs to milk-acid bacteria, and growth is fast, and adhesive power is strong, can produce lactic acid and antimicrobial substance, be widely used in the production of broiler chicken, growing and fattening pigs, piglet, milk cow etc.
Faecium (Enterococcus Faecium) is extensively present in nature and gastrointestinal tract of livestock and fowls, is a kind of beneficial microorganism, common faecium because of its characteristic such as acidproof, high temperature resistant poor, fail widespread use in fodder additives.Therefore, according to the demand of current livestock industry, screen the faecium that a strain acid-resistant and anti-high-temperature has a probiotic properties and there is vast potential for future development.
Probiotic bacterium is to feed manufacturing, especially pelletization high temperature, store transport and the tolerance such as gastrointestinal fluid and bile poor, therefore, it is possible to the viable lactic acid bacteria quantity entered in enteron aisle and kind few, and most of product exists the problems such as viable count decline on market, after fermentation, coating technique can solve this series of problem well.Also there are problems in probiotics technology of preparing, as product standard disunity, probiotic bacterium too high levels is too low affects result of use; The source of bacterial classification is failed to understand, easily degenerates, active low; Bacterial classification compatibility, synergetic property is poor; Zymotechnique is chaotic.
Summary of the invention
The object of this invention is to provide a kind of novel pig probiotic agent and preparation method thereof.
In order to realize the object of the invention, the preparation method of pig probiotic agent provided by the invention, comprises the following steps:
1) micro-capsule bag quilt: faecium (Enterococcus faecium) HEW-A588 active bacteria mud, Bacillus licheniformis (Bacillus licheniformis) active bacteria mud, yeast active bacteria mud and protection liquid are added in stirred pot successively by the weight ratio of 0.2-1.0:1.4-3.0:0.1-0.8:1, mixing speed 20-50r/m, add water after 30-60min and regulate humidity of materials to be 25-35%, obtaining micro-capsule bacterium wet-milling;
2) granulating coated: the weight ratio of micro-capsule bacterium wet-milling and soybean protein isolate being pressed 1:0.05-0.2 drops in nodulizer, regulates granularity to be 20-40 order (preferably 30 orders), obtains particle shape work in-process;
3) enteric coating: by particle shape work in-process, to be fed in cyclone fluidized bed granulating coated machine dry, dry inlet temperature controls at 60 DEG C ± 5 DEG C, and air outlet temperature controls at 32 DEG C ± 5 DEG C, time of drying 45-75min; Then carry out bag to be processed: start side spray coating device, inlet temperature controls at 40-70 DEG C, atomizing pressure is 0.1-0.2MPa, coating agent solution is sprayed to the material of eddy flow state with the speed of 20-40mL/min, form dressing rete on particulate material surface, obtain 20-50 order pig probiotic agent product through 20-70min.
Wherein, step 1) in protection liquid formula be: glycerine 2-4%, maltodextrin 1-3%, trehalose 2-4%, W-Gum 30-50%, Microcrystalline Cellulose 5-10%, polyvinylpyrrolidone 0.1-10%, vegetables oil 1-5%, surplus is water.
Step 3) in coating agent solution formula be: sodium alginate 2-10%, Xylo-Mucine 4-8%, Vltra tears 1-5%, pectin 2-7%, carrageenin 1-4%, dextran 1-10%, Oligomeric maltose 1-10%, skim-milk 1-20%, surplus is water.
Faecium (Enterococcus faecium) HEW-A588 related in the present invention is separated to obtain milk-acid bacteria from sodium selenite rectal contents, through colony morphological observation, produce acid and bacteriostasis property test etc. obtains aimed strain, this bacterial strain patience be strong, resistance and probiotic more remarkable.This bacterium has now been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC), No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, postcode 100101, deposit number CGMCC No.10547, preservation date on February 9th, 2015.
Aforesaid method, the preparation method of faecium HEW-A588 active bacteria mud is as follows:
Get the faecium HEW-A588 glycerine pipe of preservation in liquid nitrogen, after instant in 37 DEG C of water-baths, by the flat lining out separation and purification of MRS, cultivate 24-48h for 37 DEG C; On sterilisable chamber flat board, the eugonic single bacterium colony of picking, is inoculated in fresh MRS inclined-plane, cultivates 12-18h for 37 DEG C; Respectively add 2.0mL sterile saline at sterilisable chamber by test tube, with transfering loop scraping lawn, merge and make bacteria suspension; Absorption bacteria suspension 0.8mL is inoculated in and fills in the 1L triangular flask of 300mL Shake flask medium, and pH6.8,37 DEG C, 150r/min shaking culture 6.5h, obtains seed liquor.
Carry out fermentor tank pilot plant test after shake flask fermentation terminates, get 100mL shake flask fermentation seed liquor and be inoculated in 50L fermentor tank, liquid amount is 20L substratum, leavening temperature is 37 DEG C, pH value 6.8, mixing speed 120r/min, fermentation time 10h, obtains secondary seed solution; Secondary seed solution is transferred to 2 × 10
4in L fermentor tank, liquid amount 70%, then pH6.8,37 DEG C, rotating speed 120r/min ferments 10h.
Wherein, shake-flask culture based component is: sucrose 1.5%, glucose 0.5%, peptone 1.6%, yeast extract paste 0.8%, MgSO
47H
2o 0.3%, MnSO
44H
2o 0.02%, NaCl 0.5%, dibasic ammonium citrate 0.1%, CaCO
30.05%, surplus is water, pH6.8 ± 0.2.
50L fermentor tank pilot scale medium component is: caster sugar 0.5-1.0%, Trisodium Citrate 0.5-1.5%, yeast extract paste 1.0-2.0%, magnesium chloride 0.04-0.08%, sodium-chlor 0.05-0.3%, manganous sulfate 0.01-0.05%, dipotassium hydrogen phosphate 0.05-0.2%, ferric ammonium citrate 0-0.008%, tween-80 0.05-0.15%, surplus is water, pH7.0 ± 0.2.
After fermentation ends, with 12000-18000r/min centrifugal faecium HEW-A588 fermented liquid 30-50min, obtain faecium HEW-A588 active bacteria mud.
Aforesaid method, the preparation method of Bacillus licheniformis active bacteria mud is as follows:
Bacillus licheniformis is pressed the order cultivation of inclined-plane, liquid seeds, liquid fermenting; The Bacillus licheniformis of getting slant preservation is inoculated in 300mL first order seed liquid substratum, carries out shake flask fermentation cultivation, and leavening temperature is 25-45 DEG C, initial pH7.1,180r/min, and fermentation time 8-20h, obtains one-level shake-flask seed liquid; Primary seed solution is transferred to and fills in the 50L fermentor tank of 35L secondary seed medium, inoculum size is 1.5%, leavening temperature is 25-45 DEG C, initial pH value 7.1, mixing speed 140r/min, cultivate 4-12h and obtain secondary seed solution, secondary seed solution is transferred to and fills 2 × 10 of three grade fermemtation substratum
4in L fermentor tank, after inoculation, tank temperature control is at 25-45 DEG C, pH7.2, and mixing speed, at 120-180r/m, is cultivated 35-48h and obtained the lichen bacillus ferments liquid.
Wherein, slant medium is conventional beef-protein medium, and seed culture medium is glucose 1.5%, W-Gum 0.5%, dregs of beans 1.5%, yeast powder 0.5%, MgSO
47H
2o 0.05%, MnSO
44H
2o 0.05%, pH7.0 ± 0.2; Fermention medium is: molasses 2%, yeast extract paste 0.4%, analyzes starch 0.5%, NaCl 0.5%, MgSO
47H
2o 0.05%, MnSO
44H
2o 0.05%, pH7.0 ± 0.2.
After fermentation ends, with 12000-18000r/m centrifugal the lichen bacillus ferments liquid 30-50min, obtain Bacillus licheniformis active bacteria mud.
Aforesaid method, the preparation method of yeast active bacteria mud is as follows:
Pichia yeast is pressed the order cultivation of inclined-plane, liquid seeds, liquid fermenting; The Pichia yeast getting slant preservation is inoculated in 300mL first order seed liquid substratum, carries out shake flask fermentation cultivation, and leavening temperature is 25-35 DEG C, pH 6.8,120r/min, and fermentation time 10-15h, obtains one-level shake-flask seed liquid; Primary seed solution is transferred to and fills in the 50L fermentor tank of 35L secondary seed medium, inoculum size is 2%, and leavening temperature is 25-35 DEG C, pH value 6.8, mixing speed 100r/min, cultivate 8-10h and obtain second order fermentation liquid, secondary seed solution is transferred to and fills 2 × 10 of three grade fermemtation substratum
4in L fermentor tank, after inoculation, tank temperature control is at 25-35 DEG C, pH6.5-7.0, and mixing speed, at 100-150r/m, is cultivated 28-70h and obtained saccharomycetes to make fermentation liquid.
Wherein, slant medium is conventional PDA substratum, and seed culture medium is glucose 1.5%, yeast extract 1%, soy peptone 1%, NaCl 0.5%, pH6.8 ± 0.2; Fermention medium is: molasses 1.3%, yeast extract 0.7%, bean powder 1.5%, NaCl 0.5%, pH6.8 ± 0.2.
After fermentation ends, with 10000-16000r/m centrifugal saccharomycetes to make fermentation liquid 30-50min, obtain yeast active bacteria mud.
The present invention also provides the pig of being prepared by preceding method probiotic agent.
The present invention also provides described pig probiotic agent preparing the application in animal-feed.
The present invention also provides the animal-feed containing described pig probiotic agent.
The present invention further provides the application of described pig probiotic agent in poultry cultivation.
The present invention has the following advantages:
(1) pig of the present invention not only can improve the food utilization efficiency of animal with probiotic agent, cost-saving, and substantially improve the stable state of animal body digested road environment, promote absorbing of nutrition, promote growth of animal, significantly improve the production performance of animal, immune performance and reproductive performance, reduce production cost, improve economic benefit.
(2) pig of the present invention is simple with probiotic agent method for preserving, and good stability.
(3) pig of the present invention is less with probiotic agent consumption, such as, in feed, be only 10
6can remarkable effect be played during CFU/kg, be in particular in:
1, the day weight gain of test group piglet can be significantly improved, significantly reduce feedstuff-meat ratio 0.07, significantly reduce diarrhea rate.Also can improve pig house environment simultaneously, improve production performance and the economic benefit of piglet, can effective substitute antibiotics additive, be applicable to large-scale promotion and use.
2, significantly improve production performance and the immune performance of growing and fattening pigs, compared with control group, can significantly improve food consumption 5.23%, feedstuff-meat ratio significantly reduces by 0.09, and significantly reduces diarrhea rate.
3, pig probiotic agent has played certain effect to sow production performance, and controls to also play certain effect to the constipation of sow and puerperal disease.Significantly can reduce Gestation period sow and sow in lactation constipation rate (P<0.05), significantly improve health level and the autoimmunity of sow, reduce practise midwifery rate (P<0.05), and effectively reduce the incidence (P<0.05) of sow birth canal in postpartum inflammation, improve immunity of organisms, effective preventing disease, promotes intestines peristalsis, serves effective preventive effect to prevention of sow constipation, metritic incidence.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.The percentage sign " % " related in the present invention, if not specified, refers to mass percent; But the per-cent of solution, unless otherwise specified, refers to the grams containing solute in 100mL solution.
The preparation of embodiment 1 faecium HEW-A588 active bacteria mud
1.1 preparation faecium HEW-A588 fermented liquids
Get the faecium HEW-A588 glycerine pipe of preservation in liquid nitrogen, after instant in 37 DEG C of water-baths, by the flat lining out separation and purification of MRS, cultivate 24-48h for 37 DEG C; On sterilisable chamber flat board, the eugonic single bacterium colony of picking, is inoculated in fresh MRS inclined-plane, cultivates 12-18h for 37 DEG C; Respectively add 2.0mL sterile saline at sterilisable chamber by test tube, with transfering loop scraping lawn, merge and make bacteria suspension; Drawing bacteria suspension 0.8mL is inoculated in Shake flask medium (300mL/1L triangular flask), and pH6.8,37 DEG C, 150r/min shaking culture 6.5h, obtains seed liquor.
Carry out fermentor tank pilot plant test after shake flask fermentation terminates, get 100mL shake flask fermentation seed liquor and be inoculated in 50L fermentor tank, liquid amount is 20L substratum, leavening temperature is 37 DEG C, pH value 6.8, mixing speed 120r/min, fermentation time 10h, obtains secondary seed solution.Secondary seed solution is transferred to 2 × 10
4in L fermentor tank, liquid amount 70%, then pH6.8,37 DEG C, rotating speed 120r/min ferments 10h.Shake-flask culture based component is: sucrose 1.5%, glucose 0.5%, peptone 1.6%, yeast extract paste 0.8%, MgSO
47H
2o 0.3%, MnSO
44H
2o 0.02%, NaCl 0.5%, dibasic ammonium citrate 0.1%, CaCO
30.05%, surplus is water, pH6.8 ± 0.2.
50L fermentor tank pilot scale medium component is: caster sugar 0.8%, Trisodium Citrate 1.0%, yeast extract paste 1.5%, magnesium chloride 0.06%, sodium-chlor 0.1%, manganous sulfate 0.03%, dipotassium hydrogen phosphate 0.1%, ferric ammonium citrate 0.004%, tween-80 0.1%, surplus is water, pH7.0 ± 0.2.
After fermentation ends, detect fermented liquid viable count>=1.5 × 10
10cFU/mL, is about 2.0 × 10
10cFU/mL, fermented liquid is kept in refrigerator for subsequent use in 4 DEG C.
1.2 bacterium liquid are separated
The faecium HEW-A588 fermented liquid of preparation is transported in high speed tubular-bowl centrifuge by peristaltic pump and carries out the separation of bacterium liquid, the centrifugal 40min of 16000r/min, obtain faecium HEW-A588 active bacteria mud.
The preparation of embodiment 2 Bacillus licheniformis active bacteria mud
2.1 prepare the lichen bacillus ferments liquid
The order of Bacillus licheniformis (Bacillus licheniformis) by inclined-plane, liquid seeds, liquid fermenting is cultivated.The Bacillus licheniformis of getting slant preservation is inoculated in 300mL first order seed liquid substratum, carries out shake flask fermentation cultivation, and leavening temperature is 25-45 DEG C, initial pH7.1,180r/min, and fermentation time 8-20h, obtains one-level shake-flask seed liquid.Primary seed solution is transferred to (50L fermentor tank) in 35L secondary seed medium, inoculum size is 1.5%, leavening temperature is 35 DEG C, initial pH value 7.1, mixing speed 140r/min, cultivate 8h and obtain secondary seed solution, secondary seed solution to be transferred in three grade fermemtation substratum (2 × 10
4l fermentor tank), after inoculation, tank temperature control is at 35 DEG C, and pH7.2, mixing speed, at 150r/m, is cultivated 40h and obtained the lichen bacillus ferments liquid, after fermentation ends, detects fermented liquid viable count>=1.3 × 10
10cFU/mL.
Wherein slant medium is conventional beef-protein medium, and seed culture medium is glucose 1.5%, W-Gum 0.5%, dregs of beans 1.5%, yeast powder 0.5%, MgSO
47H
2o 0.05%, MnSO
44H
2o 0.05%, pH7.0 ± 0.2.Fermention medium is: molasses 2%, yeast extract paste 0.4%, analyzes starch 0.5%, NaCl 0.5%, MgSO
47H
2o 0.05%, MnSO
44H
2o 0.05%, pH7.0 ± 0.2.
2.2 bacterium liquid are separated
The lichen bacillus ferments liquid of preparation is transported to high speed tubular-bowl centrifuge by peristaltic pump, the centrifugal 40min of 16000r/m, carries out the separation of bacterium liquid, obtain Bacillus licheniformis active bacteria mud.
The preparation of embodiment 3 yeast active bacteria mud
3.1 prepare saccharomycetes to make fermentation liquid
Pichia yeast is pressed the order cultivation of inclined-plane, liquid seeds, liquid fermenting.The Pichia yeast getting slant preservation is inoculated in 300mL first order seed liquid substratum, carries out shake flask fermentation cultivation, and leavening temperature is 30 DEG C, pH 6.8,120r/min, and fermentation time 10-15h, obtains one-level shake-flask seed liquid.Primary seed solution is transferred to (50L fermentor tank) in 35L secondary seed medium, inoculum size is 2%, and leavening temperature is 30 DEG C, pH value 6.8, mixing speed 100r/min, cultivate 9h and obtain second order fermentation liquid, secondary seed solution to be transferred in three grade fermemtation substratum (2 × 10
4l fermentor tank), after inoculation, tank temperature control is at 30 DEG C, and pH6.8, mixing speed, at 120r/m, is cultivated 60h and obtained saccharomycetes to make fermentation liquid, after fermentation ends, detects fermented liquid viable count>=9.0 × 10
9cFU/mL.
Wherein slant medium is conventional PDA substratum, and seed culture medium is glucose 1.5%, yeast extract 1%, soy peptone 1%, NaCl 0.5%, pH6.8 ± 0.2.Fermention medium is: molasses 1.3%, yeast extract 0.7%, bean powder 1.5%, NaCl 0.5%, pH6.8 ± 0.2.
3.2 bacterium liquid are separated
The saccharomycetes to make fermentation liquid of preparation is transported to high speed tubular-bowl centrifuge by peristaltic pump, the centrifugal 40min of 13000r/m, carries out the separation of bacterium liquid, obtain yeast active bacteria mud.
The preparation of embodiment 4 pig probiotic agent
4.1 micro-capsule bag quilts
The preparation of protection liquid: glycerine 3%, maltodextrin 2%, trehalose 3%, W-Gum 40%, Microcrystalline Cellulose 8%, polyvinylpyrrolidone 5%, vegetables oil 3%, surplus is water, fully stirs and makes it dissolve.
Faecium HEW-A588 active bacteria mud, Bacillus licheniformis active bacteria mud, yeast active bacteria mud and protection liquid are added in stirred pot successively by the weight ratio of 0.6:2.2:0.5:1, mixing speed 50r/m, add water after 40min and regulate humidity of materials, controlling 30%; Obtain micro-capsule bacterium wet-milling, stand-by.
4.2 granulating coated
The weight ratio of the 4.1 micro-capsule bacterium wet-millings prepared and soybean protein isolate being pressed 1:0.1 drops in nodulizer, regulates granularity to be about 30 orders, obtains particle shape work in-process, stand-by.
4.3 enteric coating
By the particle shape work in-process that 4.2 prepare, be fed in cyclone fluidized bed granulating coated machine dry.Dry inlet temperature controls at about 60 DEG C, and air outlet temperature controls within 32 DEG C, time of drying 60min;
Coating agent solution is: sodium alginate 6%, Xylo-Mucine 6%, Vltra tears 3%, pectin 5%, carrageenin 3%, dextran 6%, Oligomeric maltose 6%, skim-milk 10%, and surplus is water;
Bag is processed: start side spray coating device, inlet temperature controls at 50 DEG C, and atomizing pressure is 0.2MPa, divides three shower nozzles to spray to the material of eddy flow state coating agent solution with 30mL/min speed, forms dressing rete on particulate material surface.40 order faecium HEW-A588 pig probiotic agent products are obtained through 50min.
The preparation of embodiment 5 pig probiotic agent
5.1 micro-capsule bag quilts
The preparation of protection liquid: glycerine 2%, maltodextrin 1%, trehalose 2%, W-Gum 30%, Microcrystalline Cellulose 5%, polyvinylpyrrolidone 0.1%, vegetables oil 1%, surplus is water, fully stirs and makes it dissolve.
Faecium HEW-A588 active bacteria mud, Bacillus licheniformis active bacteria mud, yeast active bacteria mud and protection liquid are added in stirred pot successively by the weight ratio of 0.2:1.4:0.1:1, mixing speed 20r/m, add water after 30min and regulate humidity of materials, controlling 25%; Obtain micro-capsule bacterium wet-milling, stand-by.
5.2 granulating coated
The weight ratio of the 5.1 micro-capsule bacterium wet-millings prepared and soybean protein isolate being pressed 1:0.05 drops in nodulizer, regulates granularity to be about 30 orders, obtains particle shape work in-process, stand-by.
5.3 enteric coating
By the particle shape work in-process that 5.2 prepare, be fed in cyclone fluidized bed granulating coated machine dry.Dry inlet temperature controls at about 60 DEG C, and air outlet temperature controls within 32 DEG C, time of drying 45min.
Coating agent solution is: sodium alginate 2%, Xylo-Mucine 4%, Vltra tears 1%, pectin 2%, carrageenin 1%, dextran 1%, Oligomeric maltose 1%, skim-milk 1%, and surplus is water;
Bag is processed: start side spray coating device, inlet temperature controls at 40 DEG C, and atomizing pressure is 0.1MPa, divides three shower nozzles to spray to the material of eddy flow state coating agent solution with 20mL/min speed, forms dressing rete on particulate material surface.20 order pig probiotic agent products are obtained through 20min.
The preparation of embodiment 6 pig probiotic agent
6.1 micro-capsule bag quilts
The preparation of protection liquid: glycerine 4%, maltodextrin 3%, trehalose 4%, W-Gum 50%, Microcrystalline Cellulose 10%, polyvinylpyrrolidone 10%, vegetables oil 5%, surplus is water, fully stirs and makes it dissolve.
Faecium HEW-A588 active bacteria mud, Bacillus licheniformis active bacteria mud, yeast active bacteria mud and protection liquid are added in stirred pot successively by the weight ratio of 1.0:3.0:0.8:1, mixing speed 50r/m, add water after 60min and regulate humidity of materials, controlling 35%; Obtain micro-capsule bacterium wet-milling, stand-by.
6.2 granulating coated
The weight ratio of the 6.1 micro-capsule bacterium wet-millings prepared and soybean protein isolate being pressed 1:0.2 drops in nodulizer, regulates granularity to be about 30 orders, obtains particle shape work in-process, stand-by.
6.3 enteric coating
By the particle shape work in-process that 6.2 prepare, be fed in cyclone fluidized bed granulating coated machine dry.Dry inlet temperature controls at about 60 DEG C, and air outlet temperature controls within 32 DEG C, time of drying 75min.
Coating agent solution is: sodium alginate 10%, Xylo-Mucine 8%, Vltra tears 5%, pectin 7%, carrageenin 4%, dextran 10%, Oligomeric maltose 10%, skim-milk 20%, and surplus is water;
Bag is processed: start side spray coating device, inlet temperature controls at 70 DEG C, and atomizing pressure is 0.2MPa, divides three shower nozzles to spray to the material of eddy flow state coating agent solution with 40mL/min speed, forms dressing rete on particulate material surface.50 order pig probiotic agent products are obtained through 70min.
The stability test of embodiment 7 pig probiotic agent
After test is chosen pig probiotic agent product prepared by embodiment 4 respectively January, 3 months, 6 months, 9 months, 12 months is preserved in sealing at normal temperatures, detect viable count of lactobacillus, measure milk-acid bacteria retention ratio compared with initial viable count of lactobacillus, and observe product characteristics, the results are shown in Table 1.
Table 1 normal temperature preserves the impact on faecium HEW-A588 survival rate
Shelf time | Viable bacteria content CFU/g | Survival rate |
0 month | 1.02×10 10 | 100% |
3 months | 1.01×10 10 | 99% |
6 months | 0.99×10 10 | 97% |
9 months | 0.98×10 10 | 96% |
12 months | 0.96×10 10 | 94% |
As shown in Table 1, pig is with probiotic agent after January, 3 months, 6 months, 9 months, 12 months are preserved in sealing, and product characteristics are good, and its bacterium survival rate all >=94%, show that pig probiotic agent stability is very good.
Embodiment 8 Growth Performance of Weaning Piglets is tested
Same parity, body weight close (Duroc × length in vain × great Bai) three way cross weanling pig (male and female half and half is selected in test, wean in 28 days) 120, be divided at random two groups (control group and test group), often process 6 hurdles, 10, every hurdle, carries out feeding experiment under equivalent environment.Diet (with reference to Chinese Pigs feeding standard) based on control group diet, add pig probiotic agent prepared by embodiment 4 in test group diet, namely on the basis of control group, feed per ton adds 100g probiotics.Raise 28 days, observe and record diarrhoea, ight soil state, the fur color and luster of piglet; The feed consumption rate of record every hurdle pig every day, statistics food consumption; Record initial individuals is heavy, end individual weight.(table 2)
The impact of probiotic agent on Growth Performance of Weaning Piglets of table 2 pig
Project | Control group | Test group |
Initial weight (kg) | 9.18±0.34 | 8.97±0.25 |
7 days heavy (kg) | 11.79±0.77 | 11.72±0.59 |
14 days heavy (kg) | 13.88±1.02 | 14.28±1.23 |
21 days heavy (kg) | 18.71±1.11 | 19.83±1.35 |
28 days heavy (kg) | 23.75±2.42 a | 24.69±2.51 b |
Total food consumption (kg) | 34.31±2.04 | 34.07±2.17 |
Feedstuff-meat ratio | 1.44±0.84 b | 1.37±0.86 a |
Diarrhoea head number | 17 | 3 |
Diarrhoea number of times | 24 | 7 |
Diarrhea rate | 24.29% | 1.25% |
Note: with the different letter representation difference of same column, wherein lowercase alphabet shows significant difference (P<0.05).
In experimentation, test group piglet fur is bright, symmetry, vivaciously active.Compared with control group, the day weight gain of test group piglet after adding pig probiotic agent in feed, can be significantly improved, significantly reduce feedstuff-meat ratio 0.07, significantly reduce diarrhea rate.(table 3)
Table 3 faecium HEW-A588 is on the impact of piglet growth environment
Project | Experimental group pig house | Negative control group pig house | Standard value |
Ammonia in Air content (mg/m 3) | 5.354±0.217 b | 8.425±0.381 a | ≤25.0 |
Air Hydrogen Sulfide content (mg/m 3) | 0.0213±0.001 b | 0.059±0.004 a | ≤10.0 |
Bacteria content (cfu/m in air 3) | 80.319±11.207 b | 48.215±12.359 a | ≤5×10 3 |
Note: with the different letter representation difference of same column, wherein lowercase alphabet shows significant difference (P<0.05).
Embodiment 9 Performance of Finishing Pigs is tested
Choose that health, about body weight 70kg, male and female ratio are consistent, (Duroc × length white × great Bai) ternary commodity growing and fattening pigs 216 that age in days is close, completely random is divided into 3 groups (positive controls, negative control group and test group), often organize 12 hurdles, 6, every hurdle pig.Negative control group: daily ration (with reference to Chinese Pigs feeding standard) based on test diet; Positive controls: test diet adds microbiotic, and namely feed per ton adds 500g duomycin on the basis of negative control group; Test group: test diet adds pig probiotic agent prepared by embodiment 4, namely on the basis of control group, feed per ton adds 100g pig probiotic agent.Test pig adopts closed colony house, and every day feeds 4 times, and free choice feeding and drinking-water, clean colony house every day at least 3 times, feeds 30 days.Feeding and management and immune programme for children identical with pig farm daily administration, regularly sterilize, find pig sick timely treatment.
Observe the mental status and the colour of skin of pig every day, test was weighed morning on the same day on an empty stomach, record charging capacity every day and surplus doses, and after off-test, m seq is weighed on an empty stomach; A situation arises to record each group of diarrhoea; Calculate daily ingestion amount, day weight gain, feed-weight ratio and diarrhea rate.(table 4)
The impact of probiotic agent on growing-finishing pigs performance of table 4 pig
Project | Negative control group | Positive controls | Test group |
Initial weight/kg | 72.57±7.96 | 72.85±8.05 | 72.88±6.91 |
End weight/kg | 96.58±11.23 | 98.21±6.57 | 98.84±8.58 |
Daily ingestion amount/(g/d) | 4488.70±54.96 | 4542.88±60.42 | 4581.14±50.72 |
Day weight gain/(g/d) | 1577.08±93.26 | 1647.50±125.16 | 1659.54±140.74 |
Feedstuff-meat ratio | 2.85±0.59 a | 2.76±0.48 b | 2.76±0.36 b |
Test head number | 72 | 72 | 72 |
Diarrhoea head number | 11 | 3 | 3 |
Diarrhea rate/% | 15.28% a | 4.17% b | 4.17% b |
Dead head number | 1 | 0 | 0 |
Skin hair color | Generally | Generally | Fur glow |
Note: with the different letter representation difference of same column, wherein lowercase alphabet shows significant difference (P<0.05).
As shown in Table 4, positive controls and test group daily ingestion amount are all significantly increased than with negative control group, and comparatively negative control group significantly improves 4.47%, 5.23% respectively; Positive controls and test group feedstuff-meat ratio respectively equal control group significantly reduce by 0.09; Compared with control group, positive controls and test group all significantly reduce diarrhea rate.In process of the test, found the pigskin hair glow of test group by the healthy vigor and growing state observing pig, the mental status is good.
Embodiment 10 sow reproductive performance is tested
Select length × large two-way cross milking sow 48 that parity is close, be in a good state of health, be divided into two groups, often organize 24,4 hurdles, 6, every hurdle, tests and within first 15 days, terminates to the wean of sucking piglets 28 age in days from farrowing, daily ration based on control group, based on test group, daily ration adds pig probiotic agent prepared by embodiment 4, and namely on the basis of control group, feed per ton adds 100g probiotics.
Because probiotics starts to feed at later pregnancy, therefore probiotics does not produce significant impact to litter size.But duration of test, test group sow significantly increases by 10.78% (P<0.05) than the average daily ingestion amount of control group, and average piglet birth weight increases 80g (P<0.05).The raising of sow production performance needs one very long period, and whole trial period is shorter, and therefore probiotics has played certain effect to sow production performance, and controls to also play certain effect to the constipation of sow and puerperal disease.Add probiotics in basal diet after, significantly can reduce Gestation period sow and sow in lactation constipation rate (P<0.05), significantly improve health level and the autoimmunity of sow, reduce practise midwifery rate (P<0.05), and effectively reduce the incidence (P<0.05) of sow birth canal in postpartum inflammation.Visible, in basal diet, add the growth and breeding that probiotics effectively can suppress pathogenic micro-organism, improve immunity of organisms, effective preventing disease, promote intestines peristalsis, effective preventive effect is served to prevention of sow constipation, metritic incidence.(table 5)
The special probiotics Z800-200 of table 5 suckling piglet is on the impact of sow reproductive performance
Project | Control group | Test group |
The average daily ingestion amount of sow (kg/ head/sky) | 5.75±0.24 a | 6.37±0.28 b |
Litter size (head/nest) | 11.87±1.27 a | 12.12±1.32 a |
Strong young number (head/nest) | 10.79±1.14 a | 11.58±1.25 b |
Average piglet Birth weight (kg/ head) | 1.29±0.11 a | 1.37±0.07 b |
Pregnant sow constipation (head) | 5 | 1 |
Pregnant sow constipation rate (%) | 20.83±1.32 a | 4.17±0.43 b |
Milking sow constipation (head) | 9 | 2 |
Milking sow constipation rate (%) | 37.50±1.22 a | 8.33±0.43 b |
Sow practise midwifery situation (head) | 8 | 3 |
Practise midwifery rate (%) | 33.33±1.87 a | 12.5±1.07 b |
Birth canal inflammation (head) | 7 | 3 |
Sickness rate (%) | 29.17±0.67 a | 12.5±0.47 b |
Note: with the different letter representation difference of same column, wherein lowercase alphabet shows significant difference (P<0.05).
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (7)
1. the preparation method of pig probiotic agent, is characterized in that, comprises the following steps:
1) micro-capsule bag quilt: faecium (Enterococcus faecium) HEW-A588 active bacteria mud, Bacillus licheniformis (Bacillus licheniformis) active bacteria mud, yeast active bacteria mud and protection liquid are added in stirred pot successively by the weight ratio of 0.2-1.0:1.4-3.0:0.1-0.8:1, mixing speed 20-50r/m, add water after 30-60min and regulate humidity of materials to be 25-35%, obtaining micro-capsule bacterium wet-milling;
2) granulating coated: the weight ratio of micro-capsule bacterium wet-milling and soybean protein isolate being pressed 1:0.05-0.2 drops in nodulizer, regulates granularity to be 20-40 order, obtains particle shape work in-process;
3) enteric coating: by particle shape work in-process, to be fed in cyclone fluidized bed granulating coated machine dry, dry inlet temperature controls at 60 DEG C ± 5 DEG C, and air outlet temperature controls at 32 DEG C ± 5 DEG C, time of drying 45-75min; Then carry out bag to be processed: start side spray coating device, inlet temperature controls at 40-70 DEG C, atomizing pressure is 0.1-0.2MPa, coating agent solution is sprayed to the material of eddy flow state with the speed of 20-40mL/min, form dressing rete on particulate material surface, obtain 20-50 order pig probiotic agent product through 20-70min;
Wherein, step 1) in protection liquid formula be: glycerine 2-4%, maltodextrin 1-3%, trehalose 2-4%, W-Gum 30-50%, Microcrystalline Cellulose 5-10%, polyvinylpyrrolidone 0.1-10%, vegetables oil 1-5%, surplus is water;
Step 3) in coating agent solution formula be: sodium alginate 2-10%, Xylo-Mucine 4-8%, Vltra tears 1-5%, pectin 2-7%, carrageenin 1-4%, dextran 1-10%, Oligomeric maltose 1-10%, skim-milk 1-20%, surplus is water.
2. preparation method according to claim 1, is characterized in that, the preparation method of described faecium HEW-A588 active bacteria mud is as follows:
Get the faecium HEW-A588 glycerine pipe of preservation in liquid nitrogen, after instant in 37 DEG C of water-baths, by the flat lining out separation and purification of MRS, cultivate 24-48h for 37 DEG C; On sterilisable chamber flat board, the eugonic single bacterium colony of picking, is inoculated in fresh MRS inclined-plane, cultivates 12-18h for 37 DEG C; Respectively add 2.0mL sterile saline at sterilisable chamber by test tube, with transfering loop scraping lawn, merge and make bacteria suspension; Absorption bacteria suspension 0.8mL is inoculated in and fills in the 1L triangular flask of 300mL Shake flask medium, pH6.8, and 37 DEG C, 150r/min shaking culture 6.5h, obtains seed liquor;
Carry out fermentor tank pilot plant test after shake flask fermentation terminates, get 100mL shake flask fermentation seed liquor and be inoculated in 50L fermentor tank, liquid amount is 20L substratum, leavening temperature is 37 DEG C, pH value 6.8, mixing speed 120r/min, fermentation time 10h, obtains secondary seed solution; Secondary seed solution is transferred to 2 × 10
4in L fermentor tank, liquid amount 70%, then pH6.8,37 DEG C, rotating speed 120r/min ferments 10h;
Wherein, shake-flask culture based component is: sucrose 1.5%, glucose 0.5%, peptone 1.6%, yeast extract paste 0.8%, MgSO
47H
2o 0.3%, MnSO
44H
2o 0.02%, NaCl 0.5%, dibasic ammonium citrate 0.1%, CaCO
30.05%, surplus is water, pH6.8 ± 0.2;
50L fermentor tank pilot scale medium component is: caster sugar 0.5-1.0%, Trisodium Citrate 0.5-1.5%, yeast extract paste 1.0-2.0%, magnesium chloride 0.04-0.08%, sodium-chlor 0.05-0.3%, manganous sulfate 0.01-0.05%, dipotassium hydrogen phosphate 0.05-0.2%, ferric ammonium citrate 0-0.008%, tween-80 0.05-0.15%, surplus is water, pH7.0 ± 0.2;
After fermentation ends, with 12000-18000r/min centrifugal faecium HEW-A588 fermented liquid 30-50min, obtain faecium HEW-A588 active bacteria mud.
3. preparation method according to claim 1, is characterized in that, the preparation method of described Bacillus licheniformis active bacteria mud is as follows:
Bacillus licheniformis is pressed the order cultivation of inclined-plane, liquid seeds, liquid fermenting; The Bacillus licheniformis of getting slant preservation is inoculated in 300mL first order seed liquid substratum, carries out shake flask fermentation cultivation, and leavening temperature is 25-45 DEG C, initial pH7.1,180r/min, and fermentation time 8-20h, obtains one-level shake-flask seed liquid; Primary seed solution is transferred to and fills in the 50L fermentor tank of 35L secondary seed medium, inoculum size is 1.5%, leavening temperature is 25-45 DEG C, initial pH value 7.1, mixing speed 140r/min, cultivate 4-12h and obtain secondary seed solution, secondary seed solution is transferred to and fills 2 × 10 of three grade fermemtation substratum
4in L fermentor tank, after inoculation, tank temperature control is at 25-45 DEG C, pH7.2, and mixing speed, at 120-180r/m, is cultivated 35-48h and obtained the lichen bacillus ferments liquid;
Wherein, slant medium is conventional beef-protein medium, and seed culture medium is glucose 1.5%, W-Gum 0.5%, dregs of beans 1.5%, yeast powder 0.5%, MgSO
47H
2o0.05%, MnSO
44H
2o 0.05%, pH7.0 ± 0.2; Fermention medium is: molasses 2%, yeast extract paste 0.4%, analyzes starch 0.5%, NaCl 0.5%, MgSO
47H
2o 0.05%, MnSO
44H
2o 0.05%, pH7.0 ± 0.2;
After fermentation ends, with 12000-18000r/m centrifugal the lichen bacillus ferments liquid 30-50min, obtain Bacillus licheniformis active bacteria mud.
4. preparation method according to claim 1, is characterized in that, the preparation method of described yeast active bacteria mud is as follows:
Pichia yeast is pressed the order cultivation of inclined-plane, liquid seeds, liquid fermenting; The Pichia yeast getting slant preservation is inoculated in 300mL first order seed liquid substratum, carries out shake flask fermentation cultivation, and leavening temperature is 25-35 DEG C, pH 6.8,120r/min, and fermentation time 10-15h, obtains one-level shake-flask seed liquid; Primary seed solution is transferred to and fills in the 50L fermentor tank of 35L secondary seed medium, inoculum size is 2%, and leavening temperature is 25-35 DEG C, pH value 6.8, mixing speed 100r/min, cultivate 8-10h and obtain second order fermentation liquid, secondary seed solution is transferred to and fills 2 × 10 of three grade fermemtation substratum
4in L fermentor tank, after inoculation, tank temperature control is at 25-35 DEG C, pH6.5-7.0, and mixing speed, at 100-150r/m, is cultivated 28-70h and obtained saccharomycetes to make fermentation liquid;
Wherein, slant medium is conventional PDA substratum, and seed culture medium is glucose 1.5%, yeast extract 1%, soy peptone 1%, NaCl 0.5%, pH6.8 ± 0.2; Fermention medium is: molasses 1.3%, yeast extract 0.7%, bean powder 1.5%, NaCl 0.5%, pH6.8 ± 0.2;
After fermentation ends, with 10000-16000r/m centrifugal saccharomycetes to make fermentation liquid 30-50min, obtain yeast active bacteria mud.
5. the pig probiotic agent prepared by method described in any one of claim 1-4.
6. the probiotic agent of pig described in claim 5 is preparing the application in animal-feed.
7. the animal-feed containing pig probiotic agent described in claim 5.
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