CN102220261B - Preparation and use of bacillus subtilis and clostridium butyricum composite bacterial preparation - Google Patents
Preparation and use of bacillus subtilis and clostridium butyricum composite bacterial preparation Download PDFInfo
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- 235000014469 Bacillus subtilis Nutrition 0.000 title abstract description 9
- 241000193171 Clostridium butyricum Species 0.000 title abstract description 6
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Abstract
The invention discloses a method for preparing a bacillus subtilis and clostridium butyricum composite bacterial preparation, which is characterized by comprising the following steps: (1) preparing seed culture solution, namely preparing a seed solution containing clostridium butyricum and a seed solution containing bacillus subtilis respectively, wherein the concentration of the bacterial solution is 0.5*10<8> to 1.0*10<8>CFU/ml; (2) preparing a coculture medium; (3) adding the seed solution of the bacillus subtilis into the coculture medium prepared by the step (1) according to a ratio of 2percent, automatically regulating the pH value to 6.5 to 7.0 by using 10-percent diluted hydrochloric acid or 10-percent sodium hydroxide, introducing gas, stirring, controlling dissolve oxygen content to be 20 to 50 percent, culturing for 18 to 20 hours at 37 DEG C, stopping introducing gas, and continuing to stir and culture for 2 to 4 hours; and (4) adding the seed solution of the clostridium butyricum prepared by the step (1) into the coculutre medium by using a peristaltic pump according a ratio of 2 percent, standing at 35 DEG C and culturing for 24 to 28 hours to obtain the bacillus subtilis and clostridium butyricum composite bacterial preparation. In the invention, a microecological preparation is mixed with feed according to a ratio of 0.5 percent, the feed is directly used to feed livestock, the diarrhea rate and death rate of the livestock are lowered effectively, yield is increased, and medicine administration cost is saved.
Description
Technical field
The present invention relates to the preparation of subtilis and clostridium butylicum composite bacteria preparation and the application on livestock and poultry cultivation thereof; Being specifically related to a kind of is raw material with subtilis, two kinds of bacterial classifications of clostridium butylicum; Utilize the two conspiracy relation during the fermentation, adopt disposable fermentation to obtain the combined prepn process and the application on the prevention livestock and poultry diarrhea thereof of subtilis and clostridium butylicum composite probiotics preparations.
Background technology
Bacillus subtilis Pseudomonas Gram-positive aerobic bacteria consumes rapidly the free oxygen in the environment in process of growth, cause the enteron aisle hypoxemia, promotes useful anerobes growth, and produces organic acid such as lactic acid, reduces the enteron aisle pH value, suppresses other pathogenic bacterium growth indirectly; In addition, subtilis thalline self synthesizes enzymes such as AMS, proteolytic enzyme, lypase, cellulase, in digestive tube, together plays a role with the intravital digestive enzymes of machine.Bibliographical information; The subtilis spore can bring back to life in enteron aisle; Oxygen in the process of growth mass consumption surrounding environment promotes the growth of probiotic bacterium (milk-acid bacteria, bifidus bacillus and clostridium butylicum etc.) in the enteron aisle, suppresses conditionality pathogenic bacterias such as intestinal bacteria, Salmonellas simultaneously.Patent 201010287464.7 discloses a kind of compound micro-ecological preparation that suppresses the pathogenic colon bacillus growth and preparation method thereof, and this method is that subtilis, butter bacillus and unusual debaryomyces hansenii bacterium are cultivated respectively, mixes by a certain percentage then.The shortcoming of this method is that the fermention medium of three kinds of bacterium needs sterilization separately, has increased energy consumption, and has reduced usage ratio of equipment.Because to the difference of fermenting process to the oxygen demand, high-efficient culture is difficult to realize when causing subtilis and anaerobism probiotic bacterium (like lactobacillus spp, bifidus bacillus and clostridium butylicum etc.).Patent 200510136003.9 discloses the preparation method of a kind of composite bacillus subtilis and lactic acid bacteria microbe formulation, and this method places the little aerobic fermentation of associating substratum simultaneously with subtilis and lactic acid bacteria culturers, though this method is cultivated when having realized aerobic and anerobes; Subtilis is an aerobic bacteria but shortcoming is; And milk-acid bacteria is an anerobes, little aerobic all be non-optimum regime to the two, be unfavorable for the increment of bacterium; Influence the bacteria content of the finished product; Reduced production efficiency, and two kinds of mikrobes cultivate simultaneously, both are restive in the quantity separately of the finished product.
Clostridium butylicum is the Gram-positive anaerobic bacillus(cillus anaerobicus); Clostridium butylicum is to regulate intestinal microecology equilibrated probiotics; Its main biological nature shows as: 1. promote the propagation and the growth of intestinal beneficial bacterium crowd (bifidus bacillus, lactobacillus spp), suppress to be harmful in the enteron aisle growth, the breeding of bacterium and spoilage organism; The correction intestinal microflora is disorderly, reduces the generation of enterotoxin; 2. in enteron aisle, can produce materials such as B vitamin, vitamin K, with health role to body; 3. the main metabolites butyric acid of clostridium butylicum is the histiocytic regeneration of gut epithelium and repairs main nutrient matter, improves the livestock and poultry feed digestibility; 4. clostridium butylicum is an anaerobic spore-bearing bacilli, and good stability is not influenced by hydrochloric acid in gastric juice, bile acide etc. in body,, under external (gemma state) room temperature, can preserve more than 3 years.The clostridium butylicum preparation is used as veterinary medicament, is used for treating the disorder of animal and bird intestines flora, increases productivity, and has obtained good effect.
Disclose at present the cultural method of multiple clostridium butylicum, still, these methods exist or production cost is high, equipment requirements is high, perhaps the production cycle long, problem such as the spore transformation efficiency is low.At first; Because clostridium butylicum is a kind of anerobes; Most working methods all need be carried out in the anaerobic environment that Yellow Protopet 2A, nitrogen or carbonic acid gas are kept; The plant and instrument complex and expensive, patent 200610125586.X discloses a kind of working method of clostridium butyricum active bacteria agent, and substratum need pass through autoclaving after Yellow Protopet 2A was kept anaerobic environment 45-60 hour.It is culture medium culturing Lactobacterium acidophilum, enterococcus faecalis and the clostridium butylicum of staple that patent 200410068185.6 discloses with peptone, yeast extract powder and glucose; After mixing, process fodder additives and be used for livestock and poultry cultivation; Its substratum consumption is big, and cost is expensive.200610107225.2 pairs of clostridium butylicums of patent and milk-acid bacteria associating culture condition are studied; This technology is that the seed culture fluid of cultivating respectively is added to co-cultivation in the associating substratum; Because two kinds of mikrobes that relate to all are anerobess; Therefore operate fairly simplely, but adopt the main carbon source of glucose, have expensive problem equally as substratum.As previously mentioned, the associating cultural method as for clostridium butylicum and aerobic bacteria (like subtilis) rarely has report especially.
Though subtilis and clostridium butylicum at the same time in the culturing process because to the otherness of oxygen demand, cause cultivating and be difficult to realize that this kind contradictory relation but plays a role for the two is collaborative in vivo and provides the foundation.Composite bacteria preparation gets into after the digestive tube, and the subtilis consume oxygen is that clostridium butylicum and other anaerobism probiotic bacteriums provide anaerobic environment on the one hand, promotes its growth and performance function; Clostridium butylicum not only improves gi tract Ph value as general anaerobism probiotic bacterium through producing acid on the other hand, improves the micro ecology of gastrointestinal tract balance, and can be further through promoting intestinal villi to grow the digestion and absorption function that improves gastrointestinal tract of livestock and fowls.Therefore, subtilis more helps keeping the micro ecology of gastrointestinal tract of livestock and fowls balance, promotes its digestion and absorption function and improves feed digesting and absorbing rate than general probiotic bacterium microbial inoculum with the clostridium butylicum composite bacteria preparation.In addition; Subtilis and clostridium butylicum all belong to genus bacillus; Its gemma has the characteristic of high temperature resistant and extruding etc.; Therefore this composite fungus agent can tolerate high temperature and the extruding of feed high temperature granulating process etc., can directly make an addition in the granulated feed and guarantees that microbic activity is unaffected.
Summary of the invention
The objective of the invention is to overcome the deficiency that exists in the prior art (like many bacterium single culture, mix at last, repeatedly sterilization cause energy consumption to increase or many bacterium unite simultaneously to cultivate with non-top condition cause the speed of growth limited etc.) and provide a kind of with aerobic bacteria and the cultivation of anerobes combined ferment; To reach the simplification production technique; Reduce production costs, improve the fermentation total viable count, strengthen clinical result of use; Further enlarge the subtilis and the clostridium butylicum associating cultural method of use; Two bacterium combined ferment technologies of the present invention are that disposable fermentation obtains compound probiotic, repeatedly sterilize and the blended trouble in having avoided producing; Reduce the living contaminants link, reduced production cost.The bacterial strain uses therefor subtilis is from Chinese common micro-organisms culture presevation administrative center, and its deposit number is: subtilis CGMCC NO. 1.884; Clostridium butylicum from its deposit number of Chinese industrial microbial strains preservation administrative center CICC is: clostridium butylicum NO. 10350, but be not limited only to this bacterial strain.
Another object of the present invention provides a kind of subtilis and clostridium butylicum composite bacteria preparation that has important application in its feeding field.
The purpose of invention is achieved in that
Concrete technology of the present invention is following:
The first step is made the seed culture fluid of subtilis and clostridium butylicum respectively, and wherein bacterial concentration is 0.5 * 10
8-1.0 * 10
8CFU/ml.
Second step; Make the associating substratum, be specially: high temperature soybean cake powder 1.8-3.5%, urea 0.1-0.3%, steeping water 0.1-0.5%, W-Gum 0.5-1.0%, potassium hydrogenphosphate 0.1-0.5%, potassium primary phosphate 0.1-0.5%, sal epsom 0.1-0.3%, manganous sulfate 0.01-0.05%.
The 3rd step joined the associating substratum with the subtilis seed liquor in 2% ratio, and the sodium hydroxide auto-control ph value of utilizing 10% Hydrogen chloride or 10% is at 6.5-7.0; Ventilation; Stir, control dissolved oxygen 20-50% cultivated 18-20 hour for 37 ℃; Stop ventilation afterwards, continued stir culture 2-4 hour.
The 4th step, utilize peristaltic pump to join in the associating substratum according to 2% ratio butyrate spindle bacillus seed liquid, leave standstill and cultivated 24-28 hour.Sampling detects total viable count, guarantees 1.0 * 10
9-5.0 * 10
9CFU/ml observes the thalli growth situation, and reaching more than 98% with the gemma rate is standard.
The 5th step, utilize tubular-bowl centrifuge centrifugal fermented liquid, add the carrier corn cob, bacterium mud is in air seasoning below 40 ℃, moisture controlled was pulverized 100 order steel sieve 5%, detects qualified after, packing is put in storage.
The 6th step, with the compound micro-ecological preparation that obtains and feed according to 0.5% mixed, the livestock and poultry of directly feeding.
The present invention has following positively effect:
1, unites cultivation and compare, only need the associating substratum is once sterilized, needn't as single culture, need sterilize respectively, so just can cut down the consumption of energy and improve the service efficiency of fermentation unit with single culture.
2, unite and cultivate fermentation of bacillus subtilis latter stage fs; Under the situation that stops to ventilate; Subtilis can exhaust oxygen remaining in the substratum rapidly, for the anaerobism clostridium butylicum provides oxygen-free environment, has practiced thrift raw material and energy consumption cost with nitrogen or carbon dioxide flooding oxygen greatly.Simultaneously, the interior aerobic processes of bacillus subtilis thalline can promote the growth and the field planting of clostridium butylicum.
3, since subtilis produce glycase during the fermentation, so we can select relatively inexpensive starch as the associating substratum carbon source, so just can reduce production costs.
4, through uniting cultivation, two strain bacterium have time enough to be suitable for, and the alternate symbiosis is convenient to two strain bacterium and is got into the collaborative afterwards performance medical functions of enteron aisle.
5, subtilis and clostridium butylicum all belong to genus bacillus; Its gemma has the characteristic of high temperature resistant and extruding etc.; Therefore this composite fungus agent can tolerate high temperature and the extruding of feed high temperature granulating process etc., can directly make an addition in the granulated feed and guarantees that microbic activity is unaffected.
The present invention has improved fermentation unit through the combined ferment technology of subtilis and clostridium butylicum, has shortened fermentation time, has reduced production cost, has strengthened its effect on livestock and poultry cultivation, makes product have stronger competitive power.
Description of drawings
Fig. 1 is a process flow sheet of the present invention.
Embodiment
Embodiment 1
1, make the seed culture fluid of subtilis and clostridium butylicum respectively, wherein bacterial concentration is 5.0 * 10
8CFU/ml.
2, make the associating substratum, be specially: high temperature soybean cake powder 1.8%, urea 0.1%, steeping water 0.1%, W-Gum 0.5%, potassium hydrogenphosphate 0.1%, potassium primary phosphate 0.1%, sal epsom 0.1%, manganous sulfate 0.01%.
3, the subtilis seed liquor in the step 1 is joined the associating substratum in 2% ratio, the sodium hydroxide auto-control ph value of utilizing 10% Hydrogen chloride or 10% is 7.0, ventilation; Stir, the control dissolved oxygen was cultivated 20 hours for 20%, 37 ℃; Stop ventilation afterwards, continued stir culture 2 hours.
4, utilize peristaltic pump to join in the associating substratum in 2% ratio the butyrate spindle bacillus seed liquid in the step 1,35 ℃ leave standstill cultivation 28 hours.Sampling detects total viable count, and total viable count is 2.70 * 10
9CFU/ml, the gemma rate is 100%.
5, utilize tubular-bowl centrifuge centrifugal fermented liquid, add the carrier corn cob, in air seasoning below 40 ℃, moisture controlled was pulverized 100 order steel sieve 5%, detects qualified after, packing is put in storage.
Embodiment 2
1, make the seed culture fluid of subtilis and clostridium butylicum respectively, wherein bacterial concentration is 1.0 * 10
8CFU/ml.
2, make the associating substratum, be specially: high temperature soybean cake powder 2.5%, urea 0.2%, steeping water 0.3%, W-Gum 0.7%, potassium hydrogenphosphate 0.3%, potassium primary phosphate 0.3%, sal epsom 0.2%, manganous sulfate 0.02%.
3, the subtilis seed liquor in the step 1 is joined the associating substratum in 2% ratio, the sodium hydroxide auto-control ph value of utilizing 10% Hydrogen chloride or 10% is 6.8, ventilation; Stir, the control dissolved oxygen was cultivated 20 hours for 30%, 37 ℃; Stop ventilation afterwards, continued stir culture 2 hours.
4, utilize peristaltic pump to join in the associating substratum in 2% ratio the butyrate spindle bacillus seed liquid in the step 1,35 ℃ leave standstill cultivation 26 hours.Sampling detects total viable count, and total viable count is 3.18 * 10
9CFU/ml, the gemma rate is 99.1%.
5, utilize tubular-bowl centrifuge centrifugal fermented liquid, add the carrier corn cob, in air seasoning below 40 ℃, moisture controlled was pulverized 100 order steel sieve 5%, detects qualified after, packing is put in storage.
Embodiment 3
1, make the seed culture fluid of subtilis and clostridium butylicum respectively, wherein bacterial concentration is 0.5 * 10
8CFU/ml.
2, make the associating substratum, be specially: high temperature soybean cake powder 3.5%, urea 0.3%, steeping water 0.5%, W-Gum 1.0%, potassium hydrogenphosphate 0.5%, potassium primary phosphate 0.5%, sal epsom 0.3%, manganous sulfate 0.05%.
3, the subtilis seed liquor in the step 1 is joined the associating substratum in 5% ratio, the sodium hydroxide auto-control ph value of utilizing 10% Hydrogen chloride or 10% is 6.5, ventilation; Stir, the control dissolved oxygen was cultivated 18 hours for 20%, 37 ℃; Stop ventilation afterwards, continued stir culture 2 hours.
4, utilize peristaltic pump to join in the associating substratum in 2% ratio the butyrate spindle bacillus seed liquid in the step 1,35 ℃ leave standstill cultivation 24 hours.Sampling detects total viable count, and total viable count is 4.36 * 10
9CFU/ml, the gemma rate is 98.6%.
5, utilize tubular-bowl centrifuge centrifugal fermented liquid, add the carrier corn cob, in air seasoning below 40 ℃, moisture controlled was pulverized 100 order steel sieve 5%, detects qualified after, packing is put in storage.
Embodiment 4
The application on prevention newborn piglet diarrhoea of subtilis and clostridium butylicum composite bacteria preparation
1, the composite bacteria preparation that embodiment 2 is obtained mixes by 0.5% with feed, directly feeding.
2, test-results
Experimental subjects is the birth piglet of certain market pig.
Experimental group: newborn piglet is fed and is added the feed of probiotics, does not carry out the prevention and control of method such as injection of antibiotic agent medication, and other feeding and management is undertaken by the ordinary method on pig farm.
Control group: diarrhoea back injection of antibiotics and other medicine take place in piglet, and combine the treatment on pig farm to manage the treatment that gives certain other drug, and daily ration, nutritive ingredient and feeding and management etc. are fully identical with experimental group.
Test-results such as table 1:
The result shows: experimental group is compared with control group, and this compound micro-ecological preparation can effectively prevent the diarrhoea of piglet, and reduces drug cost.
Embodiment 5
The application on prevention laying hen diarrhoea of subtilis and clostridium butylicum composite bacteria preparation
1, the composite bacteria preparation that embodiment 2 is obtained mixes by 0.5% with feed, directly feeding.
2, test-results
Experimental subjects is the laying hen of certain commodity egg field.
Experimental group: laying hen is fed and adds the feed of probiotics, does not carry out the prevention and control of method such as microbiotic medication, and other feeding and management is undertaken by the ordinary method of chicken house.
Control group: microbiotic and other medicine are thrown in laying hen generation diarrhoea back, and combine the treatment of chicken house to manage the treatment that gives certain other drug, and daily ration, nutritive ingredient and feeding and management etc. are fully identical with experimental group.
The result shows: experimental group laying hen and control group relatively, diarrhea rate, mortality ratio reduces significantly, and medication significantly reduces, and but obviously improves but whole opisthosoma is heavy, has increased economic benefit.
Claims (4)
1. the preparation method of subtilis and clostridium butylicum composite bacteria preparation is characterized in that it comprises the steps:
(1) makes seed culture fluid, make the kind that contains clostridium butylicum and subtilis respectively
Sub-liquid, wherein bacterial concentration is 0.5 * 10
8-1.0 * 10
8CFU/ml;
(2) make the associating substratum, be specially: high temperature soybean cake powder 1.8-3.5%, urea 0.1-0.3%, steeping water 0.1-0.5%, W-Gum 0.5-1.0%, potassium hydrogenphosphate 0.1-0.5%, potassium primary phosphate 0.1-0.5%, sal epsom 0.1-0.3%, manganous sulfate 0.01-0.05%;
(3) the subtilis seed liquor in the step (1) is joined the associating substratum in 2% ratio; The sodium hydroxide auto-control ph value of utilizing 10% Hydrogen chloride or 10% is at 6.5-7.0, and ventilation is stirred; Control dissolved oxygen 20-50%; Cultivated 18-20 hour for 37 ℃, stop ventilation afterwards, continued stir culture 2-4 hour;
(4) utilize peristaltic pump to join in the associating substratum according to 2% ratio the butyrate spindle bacillus seed liquid in the step (1), 35 ℃ leave standstill cultivation and obtained subtilis and clostridium butylicum mixed culture in 24-28 hour.
2. the preparation method of subtilis according to claim 1 and clostridium butylicum composite bacteria preparation; It is characterized in that utilizing tubular-bowl centrifuge centrifugal subtilis and clostridium butylicum mixed culture; Add the carrier corn cob; Bacterium mud is in air seasoning below 40 ℃, and moisture controlled was pulverized 100 order steel sieve and obtained subtilis and clostridium butylicum composite bacteria preparation 5%.
3. the preparation method of subtilis according to claim 1 and clostridium butylicum composite bacteria preparation, it is characterized in that step (3) cultivate to finish after, the dissolved oxygen in the associating substratum is zero, comprises that the total viable count of gemma guarantees 0.5 * 10
9-2.5 * 10
9CFU/ml.
4. the preparation method of subtilis according to claim 1 and clostridium butylicum composite bacteria preparation, it is characterized in that step (4) unite cultivate finish after, sampling detects total viable count, guarantees 1.0 * 10
9-5.0 * 10
9CFU/ml observes the thalli growth situation, changes into gemma with 98% above thalline and is advisable.
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| CN102533597A (en) * | 2011-12-23 | 2012-07-04 | 宜兴市天石饲料有限公司 | Solid state fermentation method of Clostridium butyricum |
| CN102559550B (en) * | 2011-12-30 | 2013-09-25 | 山东宝来利来生物工程股份有限公司 | Clostridium butyricum having diarrhea treating effect and application of clostridium butyricum as substitute of sodium butyrate |
| CN102960538B (en) * | 2012-12-03 | 2014-06-25 | 广西壮族自治区分析测试研究中心 | Unicellular protein feed prepared from fermented dragon fruit peel and production method of unicellular protein feed |
| CN103039698A (en) * | 2012-12-31 | 2013-04-17 | 泰山医学院 | Preparation method of bacillus subtilis powder and application thereof |
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| CN1073142C (en) * | 1994-04-21 | 2001-10-17 | 长濑生化学工业株式会社 | Bacterial preparation for agricultural use no priority right |
| US6991820B2 (en) * | 2001-07-13 | 2006-01-31 | Danisco A/S | Composition having bacteristatic and bactericidal activity against bacterial spores and vegetative cells and process for treating foods therewith |
| CN1279001C (en) * | 2005-05-30 | 2006-10-11 | 浙江大学 | Method for producing special organic fertilizer for tea plantation using fermentation of waste tea dregs |
| CN101744239A (en) * | 2008-12-01 | 2010-06-23 | 青岛东海药业有限公司 | Application of dietary fibers in the preparation of pharmaceutical preparations for preventing and curing excrement smelling or excrement smelling poisoning syndrome |
| CN101704691B (en) * | 2009-11-25 | 2012-09-26 | 中国科学院地理科学与资源研究所 | Bamboo residue organic fertilizer and method for preparing same |
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