CN102232479A - Preparation technology of bacillus feed additive agent - Google Patents
Preparation technology of bacillus feed additive agent Download PDFInfo
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- CN102232479A CN102232479A CN2010101641457A CN201010164145A CN102232479A CN 102232479 A CN102232479 A CN 102232479A CN 2010101641457 A CN2010101641457 A CN 2010101641457A CN 201010164145 A CN201010164145 A CN 201010164145A CN 102232479 A CN102232479 A CN 102232479A
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- bacillus licheniformis
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- 238000002360 preparation method Methods 0.000 title claims abstract description 33
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 27
- 238000005516 engineering process Methods 0.000 title claims abstract description 27
- 239000003674 animal food additive Substances 0.000 title claims abstract description 20
- 239000003795 chemical substances by application Substances 0.000 title abstract 5
- 241000894006 Bacteria Species 0.000 claims abstract description 19
- 230000001580 bacterial effect Effects 0.000 claims abstract description 15
- 239000000843 powder Substances 0.000 claims abstract description 13
- 238000004108 freeze drying Methods 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 239000000463 material Substances 0.000 claims abstract description 12
- 238000011081 inoculation Methods 0.000 claims abstract description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 9
- 230000004151 fermentation Effects 0.000 claims abstract description 9
- 239000000203 mixture Substances 0.000 claims abstract description 7
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 5
- 241000194108 Bacillus licheniformis Species 0.000 claims description 26
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- 239000002609 medium Substances 0.000 claims description 12
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Abstract
The invention relates to a preparation technology of a bacillus feed additive agent, belonging to the field of feed additive agent. The preparation technology comprises the following steps: 1) culturing bacillus to obtain a seed liquid; 2) carrying out liquid fermentation culture of bacillus with the inoculation amount of the seed liquid is 3-10% and until the microbial content reaches 1-2*10<10>cfu/ml; 3) collecting thalli by centrifugation; 4) freeze-drying thalli: adding a freeze-drying protectant in the collected thalli, putting the mixture in a freeze dryer for vacuum freeze-drying to obtain freeze-dried bacterial powder; and 5) adding auxiliary materials in the freeze-dried bacterial powder to obtain the bacillus feed additive agent. The feed additive agent obtained by the preparation technology has good probiotic functions of stress resistance and preventing pathogenic bacteria such as gastric acid resistance, cholate resistance, high temperature resistance, antibiotic tolerance and the like, can be used in combination with antibiotics. The prepared feed additive has high viable bacteria content, long -term storage, and obvious and stable application effects.
Description
Technical field
The present invention relates to a kind of preparation technology of bacillus feed additive, belong to feed additive field.
Background technology
As antibiotic substitute, probiotics relies on the function of the adjusting gut flora of its uniqueness, just more and more receives feed people in the industry's concern, existing in the market probiotics product.But there is the main Quality of the following aspects in the probiotics on the market: (1) viable bacteria content is low; (2) moisture is higher, and this is one of key factor that influences the probiotics quality; (3) antibiotic-resistant not; (4) to hydrochloric acid in gastric juice and cholate instability, cause still to have activity after having only the minority bacterial strain to enter enteron aisle, do not reach the required number of viable that plays a role.Since these quality problems that probiotics exists, thus its effect on animal influenced, limited the extensive use of probiotics.The microcapsules bag can be improved amount of viable bacteria in the product by method, but the valuable product that obtains by this method.Therefore, the bacterial strain that only finds a kind of existing good biological function to have very strong resistance again, simultaneously, it is simple to find out a kind of preparation technology, cost is low, but the preparation technology of the fine maintenance bacterial strain activity of energy could fundamentally solve the problem that probiotics exists.
Summary of the invention
The preparation technology who the purpose of this invention is to provide a kind of bacillus feed additive, this technology preparation process is simple, used bacterial strain has good BA, the prepared feed addictive of preparation technology of the present invention has anti-adversity such as good stomach juice-resistant, bile tolerance, high temperature resistant, tolerance common antibiotics and suppresses benefit such as pathogen and give birth to function, can with the antibiotic coupling; Product viable bacteria content height, holding time are long, and effect obviously and stable.
Inventive point of the present invention is: 1, bacillus licheniformis, and for separating, the farming oneself of big north obtains, compare with existing such bacterial strain, there are good anti-adversity and benefits such as suppressing pathogen to give birth to function; 2, preparation technology: technology is simple, makes the raising of product viable bacteria content; 3, the feed addictive that contains bacillus licheniformis of the present invention.
The objective of the invention is to be achieved through the following technical solutions:
The invention provides a kind of preparation technology of bacillus feed additive, this technology comprises the steps:
1) cultivation of bacillus licheniformis gets seed liquor; 2) liquid fermentation and culture of bacillus licheniformis, the inoculum concentration of seed liquor is 3～10%, ferments to the bacterium number and reaches 1-2 * 10
10Cfu/ml; 3) centrifugal collection thalline; 4) thalline freeze-drying: will add freeze drying protectant in the thalline of collecting, move in the freeze dryer and carry out vacuum freezedrying, become the freeze-dried vaccine powder; 5) add auxiliary material in the freeze-dried vaccine powder, get the feed addictive of bacillus.
Wherein, the used bacillus of the present invention is bacillus licheniformis Bacillus licheniformis, this bacterium separates from the healthy animal enteron aisle for big northern agricultural company, seed selection obtains, have anti-adversity such as good stomach juice-resistant, bile tolerance, high temperature resistant, tolerance common antibiotics and suppress benefit such as pathogen and give birth to function, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (Institute of Microorganism, Academia Sinica) preservation (being called for short CGMCC) on March 3rd, 2008, deposit number is CGMCC No.2384.
Used freeze dryer is a vacuum freeze drier among the present invention.
The cultivation of bacillus licheniformis of the present invention is three grades of cultivations, one-level is cultivated: with bacterial classification inoculation on the BPY plating medium, cultivate 12-24h in 30-37 ℃, make the bacillus licheniformis rejuvenation, and form single bacterium colony, picking list colony inoculation is cultivated 24-36h in 30-37 ℃ on slant medium; Secondary is cultivated: bacillus licheniformis is inoculated on the one-level BPY slant medium, in 30～37 ℃ of cultivation 12～16h, makes it be in the logarithm middle and later periods, get first order seed; Three grades of Liquid Culture: first order seed is made bacteria suspension with sterilized water, is inoculated in the BPY seed culture medium 30～37 ℃ of temperature, rotating speed 200～250rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.6～0.8 (14.4～19.2M
3/ h), cultivate 10～14h, get secondary seed solution.
In the liquid fermentation and culture process of bacillus licheniformis of the present invention, temperature is 30～37 ℃, rotating speed 220～300rpm, and tank pressure 0.05Mpa, ventilating ratio: 1: 0.6～0.8, cultivate 16～20h, to gemma formation rate more than 90%, viable count is 1～2 * 10
10Cfu/ml, then stuck fermentation gets the lichen bacillus ferments liquid.Used culture medium is: wheat bran 1～2%, dregs of beans 1～2%, sodium chloride 0.2～0.8%, magnesium sulfate 0.01～0.05%.
In the centrifugal process of bacillus licheniformis, rotating speed is controlled at below the 5000rpm.
The preparation technology of bacillus feed additive of the present invention; used freeze drying protectant is: the mixture of skimmed milk power, glycerine, sucrose, maltodextrin and sodium glutamate, according to: skimmed milk power: glycerine: sucrose: maltodextrin: the ratio of sodium glutamate=2: 0.5～1.0: 0.5～1.0: 0.5～1.5: 0.5～1.0 mixes.
Among the preparation technology of bacillus feed additive of the present invention, the auxiliary material that adds in the freeze-dried vaccine powder can be one or more the mixture in maize cob meal, defatted rice bran, corn protein powder, converted starch, the glucose.
Another object of the present invention provides the prepared feed addictive of preparation technology of this bacillus feed additive.
Preparation technology of the present invention is simple, and used lichem bacillus strain has good BA, handles 6h in manual simulation's gastric juice (pH2.0～4.0), and survival rate is more than 49.5%; Handle 6h at pig cholate solution (concentration 0.3-3g/kg), survival rate is 100%; Handle 15～30min for 50～80 ℃, survival rate is 100%; Benzyl penicillin, Amoxicillin, spectinomycin, erythromycin, kitasamycin, Bacitracin Zinc, 7 kinds of antibiosis of lincomycin are have drug resistance; Escherichia coli K88, staphylococcus aureus, three kinds of pathogenic bacteria of white diarrhea salmonella are all had good inhibitory effect, and antibacterial circle diameter is at 12～20mm; The white enzyme activity of laying eggs is 1000u/ml; The product amylase activity is: 800u/ml.
The preparation of the present invention prepared feed addictive of planting has anti-adversity such as good stomach juice-resistant, bile tolerance, high temperature resistant, tolerance common antibiotics and suppresses benefit such as pathogen and give birth to function, can with the antibiotic coupling; Product viable bacteria content height, holding time are long, and effect obviously and stable.
, it should be understood that described embodiment only is for the present invention is described, rather than limit the scope of the invention by any way more specific description the present invention by following examples.
The specific embodiment
Embodiment 1
1) bacterial classification inoculation with bacillus licheniformis activates in the BPY seed culture medium, and 37 ℃ of 200rpm cultivate 18h, obtain the gemma rate at the culture more than 95%;
2) acid resistance is measured: be inoculated in the culture of above-mentioned preparation in manual simulation's gastric juice of pH value 2.0,3.0,4.0 respectively by 5% inoculum concentration, the 0h counting compares, 2h, 6h sampling by 10 times of serial dilutions, is carried out dull and stereotyped count plate with phosphate buffer, calculates survival rate.
The preparation of manual simulation's gastric juice: measure 16.4 milliliters of 9.5%-10.5% concentrated hydrochloric acids, adding distil water to 1000 milliliter, do basic simulated gastric fluid, with hydrochloric acid or NaOH adjust pH 2.0,3.0,4.0, respectively get 10mL (9mL), be sub-packed in the test tube, 100 ℃ of following steam sterilizings 15 minutes, under aseptic condition, add the 0.100g pepsin in every 10mL liquid.
The survival rate of table 1 bacterial classification in manual simulation's gastric juice
3) bile tolerance is measured: with the culture of above-mentioned preparation, be inoculated in respectively by 5% inoculum concentration in the pig cholate solution of 0.03%, 0.1%, 0.2%, 0.3% variable concentrations, the 0h counting compares, 2h, 6h sampling is counted by 10 times of serial dilutions with physiological saline, carry out dull and stereotyped count plate, calculate survival rate.The results are shown in Table 2.
The preparation of cholate: each 9mL in 0.85% physiological saline, be sub-packed in the test tube, 121 ℃ of following steam sterilizings 30 minutes under aseptic condition, are made the pig cholate solution of 0.03%, 0.1%, 0.2%, 0.3% variable concentrations.
Table 2 bacterial classification is handled the survival rate of 6h in variable concentrations pig cholate
4) high temperature resistant mensuration:,, calculate survival rate respectively at 50 ℃, 60 ℃, 70 ℃, 80 ℃ water bath processing 15min, 30min with the culture of strains thing of above-mentioned preparation.The results are shown in Table 3.
Table 3 bacterial classification is handled the survival rate (%) of different time under different high temperature
Embodiment 2
1) cultivation of bacillus licheniformis, one-level is cultivated: bacterial classification inoculation on the BPY plating medium, in 30 cultivation 24h, is made the bacillus licheniformis rejuvenation, and forms single bacterium colony, and picking list colony inoculation is cultivated 6h in 30 ℃ on slant medium; Secondary is cultivated: bacillus licheniformis is inoculated on the one-level BPY slant medium, in 30 ℃ of about 16h of cultivation, makes it be in the logarithm middle and later periods, get first order seed; Three grades of Liquid Culture: first order seed is made bacteria suspension with sterilized water, are inoculated in the BPY seed culture medium, and 30 ℃ of temperature, rotating speed 200rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.6, cultivate 14h, get secondary seed solution and get seed liquor; 2) liquid fermentation and culture of bacillus licheniformis, the inoculum concentration of seed liquor is 3%, ferments to the bacterium number and reaches about 2 * 10
10Cfu/ml; 3) the centrifugal collection thalline of 3000rpm; 4) thalline freeze-drying: will add freeze drying protectant in the thalline of collecting: the mixture of skimmed milk power, glycerine, sucrose, maltodextrin and sodium glutamate, according to: skimmed milk power: glycerine: sucrose: maltodextrin: sodium glutamate=2: 1.0: 1.0: 1.5: 1.0 ratio mixes, move in the freeze dryer and carry out vacuum freezedrying, become the freeze-dried vaccine powder; 5) add auxiliary material glucose in the freeze-dried vaccine powder, get the feed addictive of bacillus.
Embodiment 3
1) cultivation of bacillus licheniformis, one-level is cultivated: bacterial classification inoculation on the BPY plating medium, in 37 ℃ of cultivation 12h, is made the bacillus licheniformis rejuvenation, and forms single bacterium colony, and picking list colony inoculation is cultivated 24h in 37 ℃ on slant medium; Secondary is cultivated: bacillus licheniformis is inoculated on the one-level BPY slant medium, in 37 ℃ of cultivation 12h, makes it be in the logarithm middle and later periods, get first order seed; Three grades of Liquid Culture: first order seed is made bacteria suspension with sterilized water, are inoculated in the BPY seed culture medium, and 37 ℃ of temperature, rotating speed 250rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.8, cultivate 10h, get secondary seed solution; 2) liquid fermentation and culture of bacillus licheniformis, the inoculum concentration of seed liquor is 10%, ferments to the bacterium number and reaches 1-2 * 10
10Cfu/ml; 3) the centrifugal collection thalline of 3000rpm; 4) thalline freeze-drying: will add freeze drying protectant in the thalline of collecting: the mixture of skimmed milk power, glycerine, sucrose, maltodextrin and sodium glutamate, according to: skimmed milk power: glycerine: sucrose: maltodextrin: sodium glutamate=2: 1.0: 01.0: 1.5: 1.0, move in the freeze dryer and carry out vacuum freezedrying, become the freeze-dried vaccine powder; 5) add auxiliary material maize cob meal and defatted rice bran in the freeze-dried vaccine powder, get the feed addictive of bacillus.
Experimental example 1
1, test material and method
Experimental animal and grouping: select for use 1 age in days commodity to mix strong young 360, adopt the design of completely random single-factor to divide 4 groups and test for the Chinese mugwort denapon.Establish 4 repetitions for every group, 30 of every repetitions (beginning body weight group difference is not remarkable, P＞0.05).49 days experimental periods.
Experimental design: contrast control group with virginiamycin as antibiotic: basal diet, test group 1: the feed addictive that basal diet+1 ‰ embodiment 2 make, test group II: basal diet+5mg/kg virginiamycin.
For trying daily ration and feeding and management: this experimental basis daily ration prescription and trophic level are respectively organized identical, and feeding and management condition is respectively organized in full accord, sees Table 4.
Table 4 basal diet is formed and trophic level
2, result of the test
2.1 body weight gains: the body weight gains of each test group all utmost point is significantly higher than control group (P＜0.01), test II group is the highest, but compare for 1 group with test, difference is remarkable (P＞0.05) not, two test group body weight gains improve 10.24% and 12.48% than control group respectively, illustrate that the gaining effect of each test group all is better than control group.Each stage gaining effect sees table 5 for details.
Table 5 is for examination chicken body weight statistical form unit: g
2.2 feed intake: each organizes total feed intake difference of full phase not remarkable (P＞0.05), sees table 6 for details.
Table 6 feed intake statistical form unit: gram/only
2.3 material anharmonic ratio, the death rate see Table 7
Table 7 material anharmonic ratio, mortality analysis table
Each test group material anharmonic ratio all is starkly lower than control group (P＜0.01), has reduced by 7.50% and 9.58% than control group respectively; Each test group of the death rate all significantly is lower than control group (P＜0.01), descends 75.65% and 82.90% than control group respectively; Test 1 group, the test 2 groups between difference not remarkable.
3. conclusion
3.1 adding 1 ‰ feed addictives of the present invention in the Chinese mugwort denapon meat chick daily ration can make weight gain improve 10.24%, expect that anharmonic ratio reduction by 7.50%, death rate of the onset descend 75.65%.
3.2 add 1 ‰ feed addictive groups of the present invention in the Chinese mugwort denapon meat chick daily ration and add 5mg/kg Wei Jiniya mycin group equal difference not significantly (P＞0.05) on indexs such as body weight gains, material anharmonic ratio, death rate of the onset.
Experimental example 2
Experimental animal: select the wean of 35 ages in days for use, the white x Da Bai hybridization of the length of body weight about 9kg piglet is as experimental animal, and totally 120, the test pig date of birth differs and is no more than 7 days.
Experimental design: adopt single-factor design at random.Test divides four groups, and every group has three repetitions, and each repeats 10 weanling pigs, between group and different 5% of the average weight that is no more than of the repetition mesosome method of double differences.
Test daily ration: adopt corn one dregs of beans type daily ration to make basal diet, the control group fed basal diet, test group I is the feed addictive that adds 3 preparations of 1 ‰ present embodiments on basal diet, test group II is for adding the antibiotic arsanilic acid that the 100g/T prevention is had loose bowels, the antibiotic arsanilic acid that feed addictive+interpolation 100g/T prevention have loose bowels of test group III for add 1 ‰ embodiment, 3 preparations on basal diet on basal diet.Basal diet is formed and trophic level sees Table 8.
Feeding and management: adopt ground flat to support and raise, except that the daily ration difference, other conditionally complete unanimity is carried out routinely between each group.Manually feed intake, free choice feeding, freely drink water, keep the pig house cleaning.30 days experimental periods, raise 7 days phases in advance.
Table 8 is fed, and basal diet is formed and trophic level
Test index and method: on an empty stomach pig is only weighed morning in on-test with when finishing, be respectively that unit carries out full group and weighs, calculates indexs such as feed consumption rate, daily gain, feedstuff-meat ratio, diarrhea rate with the repeating groups, and carry out otherness and significantly check.Result of the test is as follows:
(1) production performance
The production performance of test piglet sees Table 9.As can be seen from Table 9, average daily gain piglet test group I, test group II and test group III comparison according to component you can well imagine high by 7.3%, 7.5%, 9.5%; Test group I, test group II and test group III comparison reduces by 5.5%, 5.2%, 8.0% respectively according to group aspect feedstuff-meat ratio; Test group I, test group II and test group III comparison reduces by 67.3%, 47.7%, 70.1% respectively according to group aspect diarrhea rate.Test group I compares aspect daily gain, feedstuff-meat ratio difference with test group II not remarkable, but aspect prevention diarrhoea significant difference, illustrate that the feed addictive of the present invention's preparation can substitute the antibiotic that prevention is suffered from diarrhoea in the feed.Test group III is being better than test group I, test group II and control group aspect daily gain, feedstuff-meat ratio and the diarrhoea, illustrate that the feed addictive and the antibiotic of the present invention's preparation share synergy.Difference is not remarkable between each is organized aspect the average feed consumption.
The production performance of table 9 test piglet
Claims (6)
1. the preparation technology of a bacillus feed additive is characterized in that described bacillus is a bacillus licheniformis, and deposit number is CGMCC No.2384, and this technology comprises the steps:
1) cultivation of bacillus licheniformis gets seed liquor; 2) liquid fermentation and culture of bacillus licheniformis, the inoculum concentration of seed liquor is 3～10%, ferments to the bacterium number and reaches 1-2 * 10
10Cfu/ml; 3) centrifugal collection thalline; 4) thalline freeze-drying: will add freeze drying protectant in the thalline of collecting, move in the freeze dryer and carry out vacuum freezedrying, become the freeze-dried vaccine powder; 5) add auxiliary material in the freeze-dried vaccine powder, get the feed addictive of bacillus.
2. as weighing the preparation technology of 1 described bacillus feed additive, the cultivation that it is characterized in that bacillus licheniformis is three grades of cultivations, one-level is cultivated: with bacterial classification inoculation on the BPY plating medium, cultivate 12-24h in 30-37 ℃, make the bacillus licheniformis rejuvenation, and forming single bacterium colony, picking list colony inoculation is cultivated 24-36h in 30-37 ℃ on slant medium; Secondary is cultivated: bacillus licheniformis is inoculated on the one-level BPY slant medium, in 30～37 ℃ of cultivation 12～16h, makes it be in the logarithm middle and later periods, get first order seed; Three grades of Liquid Culture: first order seed is made bacteria suspension with sterilized water, is inoculated in the BPY seed culture medium 30～37 ℃ of temperature, rotating speed 200～250rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.6～0.8 (14.4～19.2M
3/ h), cultivate 10～14h, get secondary seed solution.
3. as weighing the preparation technology of 1 described bacillus feed additive, it is characterized in that in the liquid fermentation and culture process of bacillus licheniformis, 30～37 ℃ of temperature, rotating speed 220～300rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.6～0.8, cultivate 16～20h, to gemma formation rate more than 90%, viable count is 1～2 * 10
10Cfu/ml, then stuck fermentation gets the lichen bacillus ferments liquid.
4. as weighing the preparation technology of 1 described bacillus feed additive; it is characterized in that described freeze drying protectant is: the mixture of skimmed milk power, glycerine, sucrose, maltodextrin and sodium glutamate, according to: skimmed milk power: glycerine: sucrose: maltodextrin: the ratio of sodium glutamate=2: 0.5～1.0: 0.5～1.0: 0.5～1.5: 0.5～1.0 mixes.
5. as the preparation technology of power 1 described bacillus feed additive, it is characterized in that described auxiliary material comprises one or more the mixture in maize cob meal, defatted rice bran, corn protein powder, converted starch, the glucose.
One kind as power 1 to power 5 described bacillus feed additives the prepared feed addictive of preparation technology.
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| CN104605139A (en) * | 2015-01-08 | 2015-05-13 | 河北农业大学 | Bacillus subtilis-acanthopanax senticosus polysaccharide synbiotic, as well as preparation method and application thereof as feed additive |
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| CN107043724A (en) * | 2017-04-13 | 2017-08-15 | 中国农业科学院特产研究所 | A kind of bacillus licheniformis and its separation method and application |
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