CN103039698A - Preparation method of bacillus subtilis powder and application thereof - Google Patents
Preparation method of bacillus subtilis powder and application thereof Download PDFInfo
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- CN103039698A CN103039698A CN2012105839184A CN201210583918A CN103039698A CN 103039698 A CN103039698 A CN 103039698A CN 2012105839184 A CN2012105839184 A CN 2012105839184A CN 201210583918 A CN201210583918 A CN 201210583918A CN 103039698 A CN103039698 A CN 103039698A
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Abstract
The invention discloses a preparation method of bacillus subtilis powder and application thereof. The method comprises the following steps: the bacillus subtilis (capable of secreting neutral protease) with preservation number of CGMCC NO.1.892 is cultured in a soybean fluid medium, and then is subjected to shaking fermentation cultivation; after the cultivation, the cultured bacillus subtilis is centrifuged for 30 minutes at the speed of 1000 turns per minute; the precipitate is taken out and mixed with zeolite powder in weight ratio of 1:10; the mixture is oven dried for 30 minutes at the temperature of 50 DEG C; and moreover, the dried mixture is ground and screened by a 100-mesh stainless steel sieve to obtain a microecological agent, namely probiotics powder. The bacillus subtilis powder capable of producing protease can be directly used as the microecological agent (probiotics) for improving efficiency of feed utilization and reducing ammonia concentration in a colony house, or can be prepared into complete feed as per the addictive amount of 0.1% to 0.2%. According to the invention, a strain of bacillus subtilis capable of secreting protease is selected, subjected to shaking fermentation cultivation, extracted, dried and prepared into a feed level microecological agent, which can remarkably improve efficiency of feed utilization, accelerate growth of livestock, and reduce ammonia concentration of the colony house.
Description
Technical field
The present invention relates to feed additive field, be specially a kind of bacillus subtilis bacterium powder, preparation method thereof and application thereof.
Background technology
Ministry of Agriculture approved bacillus subtilis can be used as beneficial microbe (probiotic) and adds in the feed and use.The bacillus subtilis kind is a lot, and some can produce amylase, and some can produce protease.Contain a large amount of protein in chicken, the pig feed at present, a part can not be absorbed by catapepsis in these protein, causes waste, and when these protein excreted simultaneously, the generation ammonia that is decomposed was the main toxic and harmful of livestock and poultry colony house.
Summary of the invention
The present invention is directed to above weak point, a kind of bacillus subtilis bacterium powder, preparation method thereof and application thereof are provided, optimized the soybean fluid nutrient medium, the preparation method of the cultural method of bacillus subtilis and bacterium powder thereof, by testing the optimum application mode of having determined bacillus subtilis bacterium powder, by to its application, improved efficiency of feed utilization, accelerate growth of animals or poultry speed, reduced the colony house ammonia concentration.
The technical solution adopted for the present invention to solve the technical problems is: a kind of bacillus subtilis bacterium powder, preparation method thereof, use the bacillus subtilis that preserves the secretion neutral proteinase that is numbered CGMCC NO.1.892 to produce bacterial classification as bacillus subtilis, comprise the steps: that bacillus subtilis is produced bacterial classification inoculation shake flask fermentation in the soybean fluid nutrient medium to be cultivated, the main component of described soybean fluid nutrient medium is: soybean 10～30(g/L), sucrose 10～20(g/L), salt 5～10(g/L), described shake flask fermentation cultivation and fermentation temperature is 30～40 ℃, fermentation time 24～120h, pH value 6.0～8.0, rotating speed is that 120～200r/min shake flask fermentation is cultivated, after cultivation is finished, 10000 rev/mins of centrifugal 30min, get precipitation, press 1:10(W/W) mix 50 ℃ of oven dry 30min with zeolite powder, cross 100 order stainless steel sifts after grinding, namely obtain feed grade probiotic bacillus subtilis bacterium powder.
Described bacillus subtilis production bacterial classification is inoculated in the soybean fluid nutrient medium by 5～10%(V/V) to be cultivated.
Described shake flask fermentation condition of culture is: fermentation temperature is 37 ℃, fermentation time 72h, and pH value 6.5～7.5, rotating speed is 150 r/min.
The every gram of described feed grade probiotic bacillus subtilis bacterium powder bacterium powder viable bacteria content is not less than 108 CFU(CFUs).
Described bacillus subtilis bacterium powder can be used as a kind of feedstuff, mixes with other compositions of feed by 0.1%～0.2%(W/W), produces chicken, pig full-valence pellet feed.
Described bacillus subtilis bacterium powder is a kind of feed addictive----probiotic, can mix by 0.1%～0.2%(W/W) rear feeding with chicken, pig complete feed.
The invention has the beneficial effects as follows:
1, provides a kind of cultural method and bacterium powder, preparation method thereof for cultivating the bacillus subtilis that preserves the secretion neutral proteinase that is numbered CGMCC NO.1.892, in cultural method, optimized soybean Liquid Culture based formulas, this kind culture medium is suitable for the growth of this kind bacillus subtilis more, the bacterial classification survival rate is high, and culture medium raw material is easy to obtain, and production cost is low; Obtain optimum shake flask fermentation condition of culture through test, obviously improved the speed of growth and the output of bacillus subtilis mycelia; Provide a kind of bacterium powder, preparation method thereof can improve the output capacity of bacterium powder and the quality of bacterium powder.
3, the application process of this kind bacillus subtilis bacterium powder is provided, prove through zone test (pilot scale), bacillus subtilis bacterium powder can obviously improve efficiency of feed utilization as a kind of feedstuff, saves feed 3%～5%, (feedstuff-meat ratio such as the meat chick can be down to 1.75:1 from 1.81:1); Bacillus subtilis bacterium powder is as feed addictive---and feeding after-probiotic mixes with chicken, pig complete feed, accelerated growth of animals or poultry speed, daily gain can improve 3%～5%(can increase to 52g from 50g such as 30 age in days meat chick daily gains), reduce the colony house ammonia concentration, livestock and poultry colony house ammonia concentration descends, and ammonia concentration can drop to 10ul/L from 50ul/L in 50%～80%(such as the hot house broiler house).
The specific embodiment
Describe the present invention below in conjunction with specific embodiment:
Embodiment 1
(1) make soya broth: with soybean by 10%(W/V) be immersed in the water, room temperature left standstill 8-12 hour, with soy bean milk making machine the soybean of soaking is smashed, use the double gauze filter and remove residue, obtain soya-bean milk, with the soymilk concentration constant volume to 10 g/L, press sucrose 10 g/L, the ratio of salt 5g/L adds in the soya-bean milk scalding 30min with sucrose and salt, cooling, for subsequent use.
(2) inoculation: the bacillus subtilis freeze-drying lactobacillus (the bacillus subtilis freeze-drying lactobacillus that preservation used in the present invention is numbered CGMCC NO.1.892 is to buy from DSMZ of Microbe Inst., Chinese Academy of Sciences) that preservation is numbered CGMCC NO.1.892 separates in the nutrient agar panel line, 37 ℃ of cultivations of plating medium, 8～12h, transfer single bacterium colony, in the access 10ml bactericidal nurishing broth bouillon, cultivate 8～12h for 37 ℃, get zymotic fluid, in the access 250ml bactericidal nurishing broth bouillon, cultivate 8～12h for 37 ℃, produce bacterial classification as bacillus subtilis.This bacillus subtilis is produced bacterial classification by 5%(V/V) be inoculated in shake flask fermentation cultivation on the soya broth.
(3) described shake flask fermentation cultivation and fermentation temperature is 30 ℃, fermentation time 24h, pH value 6.0, rotating speed is 120r/min, after cultivation is finished, 10000 rev/mins of centrifugal 30min get precipitation, press 1:10(W/W) mix with zeolite powder, 50 ℃ of oven dry 30min, cross 100 order stainless steel sifts after grinding, be feed grade probiotic bacterium powder, the every gram of described feed grade probiotic bacterium powder bacterium powder viable bacteria content is 10
8CFU.
(4) use: following two kinds of application modes are arranged:
A. described bacillus subtilis bacterium powder can be used as a kind of feedstuff, presses 0.2%(W/W) mix with other compositions of feed, produce chicken, pig full-valence pellet feed.
B. described bacillus subtilis bacterium powder is a kind of feed addictive----probiotic, can be by 0.2%(W/W) feeding after mixing with chicken, pig complete feed.
Embodiment 2
(1) make soya broth: with soybean by 10%(W/V) be immersed in the water, room temperature left standstill 8～12 hours, with soy bean milk making machine the soybean of soaking is smashed, use the double gauze filter and remove residue, obtain soya-bean milk, with the soymilk concentration constant volume to 30 g/L, press sucrose 20 g/L, the ratio of salt 10g/L adds in the soya-bean milk scalding 30min with sucrose and salt, cooling, for subsequent use.
(2) inoculation: the bacillus subtilis freeze-drying lactobacillus that preservation is numbered CGMCC NO.1.892 separates in the nutrient agar panel line, 37 ℃ of cultivations of plating medium, 8～12h, transfer single bacterium colony, in the access 10ml bactericidal nurishing broth bouillon, cultivate 8～12h for 37 ℃, get zymotic fluid, in the access 250ml bactericidal nurishing broth bouillon, cultivate 8～12h for 37 ℃, produce bacterial classification as bacillus subtilis.This bacillus subtilis is produced bacterial classification by 10%(V/V) be inoculated in shake flask fermentation cultivation on the soya broth.
(3) described shake flask fermentation cultivation and fermentation temperature is 40 ℃, fermentation time 120h, pH value 8.0, rotating speed is 200r/min, after cultivation is finished, 10000 rev/mins of centrifugal 30min get precipitation, press 1:10(W/W) mix with zeolite powder, 50 ℃ of oven dry 30min, cross 100 order stainless steel sifts after grinding, be feed grade probiotic bacterium powder, the every gram of described feed grade probiotic bacterium powder bacterium powder viable bacteria content is 10
6CFU.
(4) use: following two kinds of application modes are arranged:
A. described bacillus subtilis bacterium powder can be used as a kind of feedstuff, presses 0.1%(W/W) mix with other compositions of feed, produce chicken, pig full-valence pellet feed.
B. described bacillus subtilis bacterium powder is a kind of feed addictive----probiotic, can be by 0.1%(W/W) feeding after mixing with chicken, pig complete feed.
Embodiment 3
(1) make soya broth: with soybean by 10%(W/V) be immersed in the water, room temperature left standstill 8-12 hour, with soy bean milk making machine the soybean of soaking is smashed, use the double gauze filter and remove residue, obtain soya-bean milk, with the soymilk concentration constant volume to 20 g/L, press sucrose 15 g/L, the ratio of salt 8g/L adds in the soya-bean milk scalding 30min with sucrose and salt, cooling, for subsequent use.
(2) inoculation: the bacillus subtilis freeze-drying lactobacillus that preservation is numbered CGMCC NO.1.892 separates in the nutrient agar panel line, 37 ℃ of cultivations of plating medium, 8～12h, transfer single bacterium colony, in the access 10ml bactericidal nurishing broth bouillon, cultivate 8～12h for 37 ℃, get zymotic fluid, in the access 250ml bactericidal nurishing broth bouillon, cultivate 8～12h for 37 ℃, produce bacterial classification as bacillus subtilis.This bacillus subtilis is produced bacterial classification by 8%(V/V) be inoculated in shake flask fermentation cultivation on the soya broth.
(3) described shake flask fermentation cultivation and fermentation temperature is 35 ℃, fermentation time 80h, pH value 7, rotating speed is 160r/min, after cultivation is finished, 10000 rev/mins of centrifugal 30min get precipitation, press 1:10(W/W) mix with zeolite powder, 50 ℃ of oven dry 30min, cross 100 order stainless steel sifts after grinding, be feed grade probiotic bacterium powder, the every gram of described feed grade probiotic bacterium powder bacterium powder viable bacteria content is not less than 10
8CFU.
(4) use: following two kinds of application modes are arranged:
A. described bacillus subtilis bacterium powder can be used as a kind of feedstuff, presses 0.15%(W/W) mix with other compositions of feed, produce chicken, pig full-valence pellet feed.
B. described bacillus subtilis bacterium powder is a kind of feed addictive----probiotic, can be by 0.15%(W/W) feeding after mixing with chicken, pig complete feed.
Embodiment 4
(1) make soya broth: with soybean by 10%(W/V) be immersed in the water, room temperature left standstill 8-12 hour, with soy bean milk making machine the soybean of soaking is smashed, use the double gauze filter and remove residue, obtain soya-bean milk, with the soymilk concentration constant volume to 10 g/L, press sucrose 10 g/L, the ratio of salt 5 g/L adds in the soya-bean milk scalding 30min with sucrose and salt, cooling, for subsequent use.
(2) inoculation: the bacillus subtilis freeze-drying lactobacillus that preservation is numbered CGMCC NO.1.892 separates in the nutrient agar panel line, 37 ℃ of cultivations of plating medium, 8～12h, transfer single bacterium colony, in the access 10ml bactericidal nurishing broth bouillon, cultivate 8～12h for 37 ℃, get zymotic fluid, in the access 250ml bactericidal nurishing broth bouillon, cultivate 8～12h for 37 ℃, produce bacterial classification as bacillus subtilis.This bacillus subtilis is produced bacterial classification by 5%(V/V) be inoculated in shake flask fermentation cultivation on the soya broth.
(3) described shake flask fermentation cultivation and fermentation temperature is 37 ℃, fermentation time 72h, pH value 6.5, rotating speed is 150r/min, after cultivation is finished, 10000 rev/mins of centrifugal 30min get precipitation, press 1:10(W/W) mix with zeolite powder, 50 ℃ of oven dry 30min, cross 100 order stainless steel sifts after grinding, be feed grade probiotic bacterium powder, the every gram of described feed grade probiotic bacterium powder bacterium powder viable bacteria content is not less than 10
8CFU.
(4) use: following two kinds of application modes are arranged:
A. described bacillus subtilis bacterium powder can be used as a kind of feedstuff, presses 0.1%(W/W) mix with other compositions of feed, produce chicken, pig full-valence pellet feed.
B. described bacillus subtilis bacterium powder is a kind of feed addictive----probiotic, can be by 0.1%(W/W) feeding after mixing with chicken, pig complete feed.
Embodiment 5
(1) make soya broth: with soybean by 10%(W/V) be immersed in the water, room temperature left standstill 8～12 hours, with soy bean milk making machine the soybean of soaking is smashed, use the double gauze filter and remove residue, obtain soya-bean milk, with the soymilk concentration constant volume to 10 g/L, press sucrose 10 g/L, the ratio of salt 5 g/L adds in the soya-bean milk scalding 30min with sucrose and salt, cooling, for subsequent use.
(2) inoculation: the bacillus subtilis freeze-drying lactobacillus that preservation is numbered CGMCC NO.1.892 separates in the nutrient agar panel line, 37 ℃ of cultivations of plating medium, 8～12h, transfer single bacterium colony, in the access 10ml bactericidal nurishing broth bouillon, cultivate 8～12h for 37 ℃, get zymotic fluid, in the access 250ml bactericidal nurishing broth bouillon, cultivate 8～12h for 37 ℃, produce bacterial classification as bacillus subtilis.This bacillus subtilis is produced bacterial classification by 5%(V/V) be inoculated in shake flask fermentation cultivation on the soya broth.
(3) described shake flask fermentation cultivation and fermentation temperature is 37 ℃, fermentation time 72h, pH value 7.5, rotating speed is 150r/min, after cultivation is finished, 10000 rev/mins of centrifugal 30min get precipitation, press 1:10(W/W) mix with zeolite powder, 50 ℃ of oven dry 30min, cross 100 order stainless steel sifts after grinding, be feed grade probiotic bacterium powder, the every gram of described feed grade probiotic bacterium powder bacterium powder viable bacteria content is not less than 10
8CFU.
(4) use: following two kinds of application modes are arranged:
A. described bacillus subtilis bacterium powder can be used as a kind of feedstuff, presses 0.1%(W/W) mix with other compositions of feed, produce chicken, pig full-valence pellet feed.
B. described bacillus subtilis bacterium powder is a kind of feed addictive----probiotic, can be by 0.1%(W/W) feeding after mixing with chicken, pig complete feed.
Certainly, above-mentioned explanation is not to be restriction to invention, the present invention also be not limited in above-mentioned for example, the variation that those skilled in the art make in essential scope of the present invention, remodeling, interpolation or replacement also should belong to protection scope of the present invention.
Claims (6)
1. bacillus subtilis bacterium powder, preparation method thereof, it is characterized in that: use the bacillus subtilis that preserves the secretion neutral proteinase that is numbered CGMCC NO.1.892 to produce bacterial classification as bacillus subtilis, comprise the steps: that bacillus subtilis is produced bacterial classification inoculation shake flask fermentation in the soybean fluid nutrient medium to be cultivated, the main component of described soybean fluid nutrient medium is: soybean 10～30(g/L), sucrose 10～20(g/L), salt 5～10(g/L), described shake flask fermentation cultivation and fermentation temperature is 30～40 ℃, fermentation time 24～120h, pH value 6.0～8.0, rotating speed is that 120～200r/min shake flask fermentation is cultivated, after cultivation is finished, 10000 rev/mins of centrifugal 30min, get precipitation, press 1:10(W/W) mix 50 ℃ of oven dry 30min with zeolite powder, cross 100 order stainless steel sifts after grinding, namely obtain feed grade probiotic bacillus subtilis bacterium powder.
2. a kind of bacillus subtilis bacterium powder, preparation method thereof according to claim 1 is characterized in that: described bacillus subtilis produces bacterial classification and is inoculated in the soybean fluid nutrient medium by 5～10%(V/V) and cultivates.
3. a kind of bacillus subtilis bacterium powder, preparation method thereof according to claim 1, it is characterized in that: described shake flask fermentation condition of culture is: fermentation temperature is 37 ℃, fermentation time 72h, pH value 6.5～7.5, rotating speed is 150 r/min.
4. a kind of bacillus subtilis bacterium powder, preparation method thereof according to claim 1, it is characterized in that: the every gram of described feed grade probiotic bacillus subtilis bacterium powder bacterium powder viable bacteria content is not less than 10
8CFU.
5. the application of the resulting bacillus subtilis bacterium of preparation method according to claim 1 powder, it is characterized in that: described bacillus subtilis bacterium powder can be used as a kind of feedstuff, mix with other compositions of feed by 0.1%～0.2%(W/W), produce chicken, pig full-valence pellet feed.
6. the according to claim 1 application of the resulting bacillus subtilis bacterium of described preparation method powder, it is characterized in that: described bacillus subtilis bacterium powder is a kind of feed addictive----probiotic, can mix by 0.1%～0.2%(W/W) rear feeding with chicken, pig complete feed.
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| CN103555640A (en) * | 2013-11-18 | 2014-02-05 | 山东蔚蓝生物科技有限公司 | Bacillus subtilis and application thereof to livestock breeding |
| CN103923855A (en) * | 2014-04-09 | 2014-07-16 | 湖北普信工业微生物应用技术开发有限公司 | Organic nitrogen source, and preparation method and application thereof |
| CN107118060A (en) * | 2017-06-29 | 2017-09-01 | 重庆市万源禽蛋食品有限公司 | Biological organic fertilizer and preparation method thereof |
| CN109170130A (en) * | 2018-08-08 | 2019-01-11 | 浙江炯炜海洋生物科技有限公司 | The method that solid state fermentation aquatic products leftover bits and pieces prepares fish meal |
| CN112662586A (en) * | 2021-01-15 | 2021-04-16 | 洪晓桑 | Probiotic culture medium and probiotic preparation |
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Cited By (9)
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| CN103555640A (en) * | 2013-11-18 | 2014-02-05 | 山东蔚蓝生物科技有限公司 | Bacillus subtilis and application thereof to livestock breeding |
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| CN109170130A (en) * | 2018-08-08 | 2019-01-11 | 浙江炯炜海洋生物科技有限公司 | The method that solid state fermentation aquatic products leftover bits and pieces prepares fish meal |
| CN109170130B (en) * | 2018-08-08 | 2022-02-08 | 浙江炯炜海洋生物科技有限公司 | Method for preparing fish meal by solid-state fermentation of aquatic product leftovers |
| CN112662586A (en) * | 2021-01-15 | 2021-04-16 | 洪晓桑 | Probiotic culture medium and probiotic preparation |
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Application publication date: 20130417 |