CN105941835A - Method for preparing bacillus subtilis traditional Chinese medicine feed additive - Google Patents
Method for preparing bacillus subtilis traditional Chinese medicine feed additive Download PDFInfo
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Abstract
The invention relates to a method for preparing a bacillus subtilis traditional Chinese medicine feed additive. The method comprises the following steps: activating bacillus subtilis for feed, and performing expanded culture so as to obtain a seed solution; performing superfine grinding on kudzu vine roots, polygonatum sibiricum and medical stone, and performing sterilization so as to obtain sterilized traditional Chinese medicine powder A; inoculating the seed solution into a culture liquid, and culturing for 8 hours so as to obtain a bacterial liquid; adding the sterilized traditional Chinese medicine powder A into the bacterial liquid, and continuously culturing so as to obtain a secondary seed solution; performing superfine grinding on dried tangerine or orange peel, houttuynia cordata and hawthorn, and performing sterilization so as to obtain sterilized traditional Chinese medicine powder B; inoculating the secondary seed solution into a liquid fermentation culture medium, and culturing so as to obtain fermentation broth; adding the sterilized traditional Chinese medicine powder B into the fermentation broth, and continuously fermenting for 10-15 hours so as to obtain final fermentation broth; performing vacuum concentration on the final fermentation broth, performing atomization, performing parallel flow drying by using hot air, leading out dried materials by using a cyclone separator, and recycling, thereby obtaining the bacillus subtilis traditional Chinese medicine feed additive. The bacillus subtilis traditional Chinese medicine feed additive is small in water content, good in bacterium activity and wide in market prospect, and the viable count is 700-1800 billion/g.
Description
Technical field
The present invention relates to the preparation method of a kind of mycopowder, the preparation method of a kind of bacillus subtilis Chinese medicine feed additive.
Background technology
Hay spore is as the conventional bacterium in a kind of microorganism formulation, after bacillus subtilis enters animal intestinal with spore form, can again sprout from resting state rapidly, as a kind of aerobe, bacillus subtilis can consume the free oxygen in digestive tract in sprouting, growth course rapidly, in large quantities, causes anaerobic environment；And harmful bacteria mostly typically is aerobic bacteria, therefore its growth is suppressed, and anaerobism profitable strain becomes dominant microflora.Simultaneously, bacillus subtilis also has stronger protease, amylase and lipase active, can be activated enzyme by internal activation of zymogen, also can secrete a series of extracellular enzyme, such as pectase, glucanase, cellulase etc., the materials such as non-starch polysaccharides(nsp) can be helped to decompose in plant feed, improve efficiency of feed utilization；And acid intestinal environment can the intestinal peristalsis promoting of stimulating animal, facilitating digestion absorbs.Additionally, bacillus subtilis itself is as the nonspecific immunity factor, thalline or cell wall constituent stimulation of host cell can be passed through, thus activating macrophage, or can be as the immunological adjuvant of nonspecific immune reaction；Also the immunoglobulin secretions such as IgA can be made to strengthen, improve Immune discrimination power；Stimulator antigen is delivery cell (APC), and inducing T cell, B cell produce cytokine, activate systemic immune system by lymph.Meanwhile, bacillus subtilis can synthesize vitamin B group, and vitamin E, K during growth and breeding, may participate in the metabolism of animal body, has the effects such as promotion calcium, phosphorus absorption, antioxidation, coagulant blood.Hay spore manufacturing enterprise in the market is a lot, but majority is enterprise, workshop-based production on a small scale, and apparatus and process falls behind, and grass bacillus cereus quality is uneven, and the composition in product constitutes not science, although some number Han bacterium is many, but miscellaneous bacteria rate is high, and effect is bad.Therefore developing the bacillus cereus production technology of a kind of High Density Cultivation and high Activity recovery, preparation has bacillus cereus mycopowder or the preparation of stabilizing active, to promoting ecological ideas cultivation, reduces antibiotic usage, improves animal food source safety particularly important.
Summary of the invention
The present invention seeks to the deficiency for solving above-mentioned technical problem, the preparation method of a kind of bacillus subtilis Chinese medicine feed additive is provided, prepared bacillus subtilis Chinese medicine feed additive water content is less than 6%, viable count is at 7000-18000 hundred million/g, additive preserves 2 years, and bacillus subtilis therein still can preserve 95% bacterial activity.
The preparation method of a kind of bacillus subtilis Chinese medicine feed additive, comprises the following steps:
Step one, feeding bacillus subtilis is seeded on inclined-plane seed culture medium, cultivates 48h for 35-37 DEG C；In described inclined-plane seed culture medium, the mass percentage content of each composition is: glucose 0.5-1.5%, peptone 0.2-2%, Carnis Bovis seu Bubali cream 0.5-1.5%, yeast extract 0.5-1.5%, sodium chloride 0.5%, agar 1.5-2%, and surplus is water；
Step 2, from cultivate fresh inclined-plane picking 2-4 ring lawn be inoculated in the 250ml triangular flask equipped with 50ml fluid enlargement culture base, 35-37 DEG C, 180-240rpm shaking table shaken cultivation 16-24h, obtain seed liquor, standby；
In described fluid enlargement culture base, the mass percentage content of each composition is: glucose 0.5-1.5%, peptone 0.2-2%, Carnis Bovis seu Bubali cream 0.5-1.5%, yeast extract 0.5-1.5%, sodium chloride 0.5%, and surplus is water；
Step 3, take Radix Puerariae, Rhizoma Polygonati and meal wheat stone according to the ratio that mass ratio is 2:1:0.2, be placed in super micron mill and carry out micronizing, cross 60-80 mesh sieve, gained Chinese medicine powder is carried out high-temperature heat sterilization, is cooled to room temperature, obtain Chinese medicine powder A after sterilizing, standby；
Step 4, the seed liquor of above-mentioned cultivation being inoculated into equipped with in the 5L fermentation tank of 3L culture fluid, inoculum concentration is the 5-15% of culture fluid volume；After inoculation terminates, in 150-250rpm, 1:1 ventilation, after cultivating 8h under the conditions of pH6.5-7.0,35-37 DEG C, obtain bacterium solution；In bacterium solution, add Chinese medicine powder A after the sterilizing of its weight 6%-7%, after stirring, continue to be 150-250rpm, 1:1 ventilation, cultivate 8-12h under the conditions of pH6.5-7.0,35-37 DEG C, obtain secondary seed solution, standby；
In described culture fluid, the mass percentage content of each composition is: glucose 0.5-1.5%, soybean cake powder 0.5-2.0%, peptone 0.5-2.5%, ammonium sulfate 0.25-1.5%, disodium hydrogen phosphate 0.05-0.20%, sodium dihydrogen phosphate 0.05-0.20%, magnesium sulfate 0.05-0.5%, calcium chloride 0.01-0.10%, surplus is water；
Step 5, take Pericarpium Citri Reticulatae, Herba Houttuyniae and Fructus Crataegi for the ratio of 5:2:1 in mass ratio, be placed in super micron mill and carry out micronizing, cross 60-80 mesh sieve, gained Chinese medicine powder is carried out high-temperature heat sterilization, is cooled to room temperature, obtain Chinese medicine powder B after sterilizing, standby；
Step 6, the secondary seed solution of cultivation being inoculated in equipped with in the 50L fermentation tank of 30-35L liquid fermentation medium, inoculum concentration is the 5-15% of fermentation medium volume；Inoculation is cultivated 20-25h by 1:0.6-0.75 ventilation, 150-220rpm, pH6.5-7.0,35-37 DEG C, is obtained fermentation liquid after terminating；Adding Chinese medicine powder B after the sterilizing of its quality 2% in fermentation liquid, in 1:0.6-0.75 ventilation, 150-220rpm, pH6.5-7.0,35-37 DEG C is continued fermentation 10-15h and obtains final fermentation liquid, standby；
In described liquid fermentation medium, the mass percentage content of each composition is: glucose 1.0-3.0%, starch 0.5-2.0%, soybean cake powder 0.5-2.0%, peptone 0.2-2%, ammonium sulfate 0.25-1.5%；Calcium carbonate 0.01-0.05%, dipotassium hydrogen phosphate 0.01-0.05%, manganese phosphate 0.01-0.05%, calcium chloride 0.01-0.05%, surplus is water；
Step 7, by in final for gained fermentation liquid transport pump to two effect vacuum concentration equipment, one effect temperature 75-80 DEG C, vacuum 0.05-0.055MPa, two effects 50-55 DEG C, vacuum 0.058-0.065MPa, cocnentration factor is 3-4:1, concentration adds starch mix homogeneously in 10-20% ratio after terminating, it is delivered to be spray-dried top of tower by material with pump, it is atomized under 15000-18000rpm by centrifugal type atomizer, carry out and fluidized drying with the hot-air that inlet temperature is 140-155 DEG C, dried material reclaims after cyclone separator is drawn from splicing bucket, i.e. prepare bacillus subtilis Chinese medicine feed additive.
Provide the benefit that:
Bacillus subtilis Chinese medicine feed additive of the present invention, feature for feeding bacillus subtilis, it is carried out classification cultivation, in conjunction with the interpolation of Chinese medicine ingredients in each step, bacterial activity can be increased, extend its cycle keeping activity, make prepared mycopowder viable count at 7000-18000 hundred million/g, preserve 2 years and still can preserve 95% bacterial activity.The combination of each step in present invention process, ensure that the raw material Chinese medicine used interacts with feeding bacillus subtilis, prepared feed additive is finally made to be applicable to various animal, including poultry, domestic animal etc., the absorption to nutrient substance of the animal the intestines and stomach can be promoted, animal health fast-growth can be promoted.Experimental verification, after feed additive of the present invention adds in feedstuff, can significantly improve the disease resistance of animal body, accelerate animal growth rate.And feed additive of the present invention is without preservative, harmless to carcass, there is wide market application foreground.
Detailed description of the invention
Embodiment
1
The preparation method of a kind of bacillus subtilis Chinese medicine feed additive, comprises the following steps:
Step one, feeding bacillus subtilis is seeded on inclined-plane seed culture medium, cultivates 48h for 35 DEG C；In described inclined-plane seed culture medium, the mass percentage content of each composition is: glucose 0.5%, peptone 0.2%, Carnis Bovis seu Bubali cream 0.5%, yeast extract 0.5%, sodium chloride 0.5%, agar 1.5%, and surplus is water；
Step 2, from cultivate fresh inclined-plane picking 2-4 ring lawn be inoculated in the 250ml triangular flask equipped with 50ml fluid enlargement culture base, 35 DEG C, 240rpm shaking table shaken cultivation 16h, obtain seed liquor, standby；
In described fluid enlargement culture base, the mass percentage content of each composition is: glucose 0.5%, peptone 0.2%, Carnis Bovis seu Bubali cream 0.5%, yeast extract 0.5%, sodium chloride 0.5%, and surplus is water；
Step 3, take Radix Puerariae, Rhizoma Polygonati and meal wheat stone according to the ratio that mass ratio is 2:1:0.2, be placed in super micron mill and carry out micronizing, cross 60 mesh sieves, gained Chinese medicine powder is carried out high-temperature heat sterilization, is cooled to room temperature, obtain Chinese medicine powder A after sterilizing, standby；
Step 4, the seed liquor of above-mentioned cultivation being inoculated into equipped with in the 5L fermentation tank of 3L culture fluid, inoculum concentration is the 5% of culture fluid volume；After inoculation terminates, in 150rpm, 1:1 ventilation, pH6.5, after cultivating 8h under the conditions of 35 DEG C, obtain bacterium solution；In bacterium solution, add Chinese medicine powder A after the sterilizing of its weight 6%, after stirring, continue to be 150rpm, 1:1 ventilation, pH6.5, cultivate 8h under the conditions of 35 DEG C, obtain secondary seed solution, standby；
In described culture fluid, the mass percentage content of each composition is: glucose 0.5%, soybean cake powder 0.5%, peptone 0.5%, ammonium sulfate 0.25%, disodium hydrogen phosphate 0.05%, sodium dihydrogen phosphate 0.05%, magnesium sulfate 0.05%, calcium chloride 0.01%, and surplus is water；
Step 5, take Pericarpium Citri Reticulatae, Herba Houttuyniae and Fructus Crataegi for the ratio of 5:2:1 in mass ratio, be placed in super micron mill and carry out micronizing, cross 60 mesh sieves, gained Chinese medicine powder is carried out high-temperature heat sterilization, is cooled to room temperature, obtain Chinese medicine powder B after sterilizing, standby；
Step 6, the secondary seed solution of cultivation being inoculated in equipped with in the 50L fermentation tank of 30L liquid fermentation medium, inoculum concentration is the 5% of fermentation medium volume；Inoculate after terminating by 1:0.6 ventilation, 150rpm, pH6.5, cultivate 20h, obtain fermentation liquid for 35 DEG C；Adding Chinese medicine powder B after the sterilizing of its quality 2% in fermentation liquid, at 1:0.6 ventilation, 150rpm, pH6.5,35 DEG C are continued fermentation 10h and obtain final fermentation liquid, standby；
In described liquid fermentation medium, the mass percentage content of each composition is: glucose 1.0%, starch 0.5%, soybean cake powder 0.5%, peptone 0.2%, ammonium sulfate 0.25%；Calcium carbonate 0.01%, dipotassium hydrogen phosphate 0.01%, manganese phosphate 0.01%, calcium chloride 0.01%, surplus is water；
Step 7, by final for gained fermentation liquid transport pump to two effect vacuum concentration equipment in, one effect temperature 75 DEG C, vacuum 0.05MPa, two effects 50 DEG C, vacuum 0.058MPa, cocnentration factor is 3:1, concentration adds starch mix homogeneously in 10% ratio after terminating, it is delivered to be spray-dried top of tower by material with pump, it is atomized under 15000rpm by centrifugal type atomizer, carrying out and fluidized drying with the hot-air that inlet temperature is 140 DEG C, dried material reclaims after cyclone separator is drawn from splicing bucket, i.e. prepares bacillus subtilis Chinese medicine feed additive.Prepared bacillus subtilis Chinese medicine feed additive water content is less than 6%, and viable count preserves 2 years at 700,000,000,000/g, additive, and bacillus subtilis therein still can preserve 95% bacterial activity.
Feeding bacillus subtilis described in the present embodiment is bought from Beijing North Na Chuanlian Bioteknologisk Institut, and bacterium numbering is BNCC189983.
Embodiment
2
The preparation method of a kind of bacillus subtilis Chinese medicine feed additive, comprises the following steps:
Step one, feeding bacillus subtilis is seeded on inclined-plane seed culture medium, cultivates 48h for 37 DEG C；In described inclined-plane seed culture medium, the mass percentage content of each composition is: glucose 1.5%, peptone 2%, Carnis Bovis seu Bubali cream 1.5%, yeast extract 1.5%, sodium chloride 0.5%, agar 2%, and surplus is water；
Step 2, from cultivate fresh inclined-plane picking 2-4 ring lawn be inoculated in the 250ml triangular flask equipped with 50ml fluid enlargement culture base, 37 DEG C, 240rpm shaking table shaken cultivation 24h, obtain seed liquor, standby；
In described fluid enlargement culture base, the mass percentage content of each composition is: glucose 1.5%, peptone 2%, Carnis Bovis seu Bubali cream 1.5%, yeast extract 1.5%, sodium chloride 0.5%, and surplus is water；
Step 3, take Radix Puerariae, Rhizoma Polygonati and meal wheat stone according to the ratio that mass ratio is 2:1:0.2, be placed in super micron mill and carry out micronizing, cross 80 mesh sieves, gained Chinese medicine powder is carried out high-temperature heat sterilization, is cooled to room temperature, obtain Chinese medicine powder A after sterilizing, standby；
Step 4, the seed liquor of above-mentioned cultivation being inoculated into equipped with in the 5L fermentation tank of 3L culture fluid, inoculum concentration is the 15% of culture fluid volume；After inoculation terminates, in 250rpm, 1:1 ventilation, pH7.0, after cultivating 8h under the conditions of 37 DEG C, obtain bacterium solution；In bacterium solution, add Chinese medicine powder A after the sterilizing of its weight 7%, after stirring, continue to be 250rpm, 1:1 ventilation, pH7.0, cultivate 12h under the conditions of 37 DEG C, obtain secondary seed solution, standby；
In described culture fluid, the mass percentage content of each composition is: glucose 1.5%, soybean cake powder 2.0%, peptone 2.5%, ammonium sulfate 1.5%, disodium hydrogen phosphate 0.20%, sodium dihydrogen phosphate 0.20%, magnesium sulfate 0.5%, calcium chloride 0.10%, and surplus is water；
Step 5, take Pericarpium Citri Reticulatae, Herba Houttuyniae and Fructus Crataegi for the ratio of 5:2:1 in mass ratio, be placed in super micron mill and carry out micronizing, cross 80 mesh sieves, gained Chinese medicine powder is carried out high-temperature heat sterilization, is cooled to room temperature, obtain Chinese medicine powder B after sterilizing, standby；
Step 6, the secondary seed solution of cultivation being inoculated in equipped with in the 50L fermentation tank of 35L liquid fermentation medium, inoculum concentration is the 15% of fermentation medium volume；Inoculate after terminating by 1:0.75 ventilation, 220rpm, pH7.0, cultivate 25h, obtain fermentation liquid for 37 DEG C；Adding Chinese medicine powder B after the sterilizing of its quality 2% in fermentation liquid, at 1:0.75 ventilation, 220rpm, pH7.0,37 DEG C are continued fermentation 15h and obtain final fermentation liquid, standby；
In described liquid fermentation medium, the mass percentage content of each composition is: glucose 3.0%, starch 2.0%, soybean cake powder 2.0%, peptone 2%, ammonium sulfate 1.5%；Calcium carbonate 0.05%, dipotassium hydrogen phosphate 0.05%, manganese phosphate 0.05%, calcium chloride 0.05%, surplus is water；
Step 7, by final for gained fermentation liquid transport pump to two effect vacuum concentration equipment in, one effect temperature 80 DEG C, vacuum 0.055MPa, two effects 55 DEG C, vacuum 0.065MPa, cocnentration factor is 4:1, concentration adds starch mix homogeneously in 20% ratio after terminating, it is delivered to be spray-dried top of tower by material with pump, it is atomized under 18000rpm by centrifugal type atomizer, carrying out and fluidized drying with the hot-air that inlet temperature is 155 DEG C, dried material reclaims after cyclone separator is drawn from splicing bucket, i.e. prepares bacillus subtilis Chinese medicine feed additive.Prepared bacillus subtilis Chinese medicine feed additive water content is less than 6%, and viable count preserves 2 years at 1,200,000,000,000/g, additive, and bacillus subtilis therein still can preserve 95% bacterial activity.
Embodiment
3
The preparation method of a kind of bacillus subtilis Chinese medicine feed additive, comprises the following steps:
Step one, feeding bacillus subtilis is seeded on inclined-plane seed culture medium, cultivates 48h for 36 DEG C；In described inclined-plane seed culture medium, the mass percentage content of each composition is: glucose 1.0%, peptone 1.5%, Carnis Bovis seu Bubali cream 1.0%, yeast extract 1.2%, sodium chloride 0.5%, agar 1.8%, and surplus is water；
Step 2, from cultivate fresh inclined-plane picking 3 ring lawn be inoculated in the 250ml triangular flask equipped with 50ml fluid enlargement culture base, 36 DEG C, 200rpm shaking table shaken cultivation 20h, obtain seed liquor, standby；
In described fluid enlargement culture base, the mass percentage content of each composition is: glucose 1.2%, peptone 1.6%, Carnis Bovis seu Bubali cream 1.2%, yeast extract 1.2%, sodium chloride 0.5%, and surplus is water；
Step 3, take Radix Puerariae, Rhizoma Polygonati and meal wheat stone according to the ratio that mass ratio is 2:1:0.2, be placed in super micron mill and carry out micronizing, cross 70 mesh sieves, gained Chinese medicine powder is carried out high-temperature heat sterilization, is cooled to room temperature, obtain Chinese medicine powder A after sterilizing, standby；
Step 4, the seed liquor of above-mentioned cultivation being inoculated into equipped with in the 5L fermentation tank of 3L culture fluid, inoculum concentration is the 10% of culture fluid volume；After inoculation terminates, in 200rpm, 1:1 ventilation, after cultivating 8h under the conditions of pH6.5-7.0,35-37 DEG C, obtain bacterium solution；In bacterium solution, add Chinese medicine powder A after the sterilizing of its weight 6.5%, after stirring, continue to be 200rpm, 1:1 ventilation, pH6.8, cultivate 10h under the conditions of 36 DEG C, obtain secondary seed solution, standby；
In described culture fluid, the mass percentage content of each composition is: glucose 1.0%, soybean cake powder 1.5%, peptone 1.3%, ammonium sulfate 1.3%, disodium hydrogen phosphate 0.15%, sodium dihydrogen phosphate 0.15%, magnesium sulfate 0.35%, calcium chloride 0.05%, and surplus is water；
Step 5, take Pericarpium Citri Reticulatae, Herba Houttuyniae and Fructus Crataegi for the ratio of 5:2:1 in mass ratio, be placed in super micron mill and carry out micronizing, cross 70 mesh sieves, gained Chinese medicine powder is carried out high-temperature heat sterilization, is cooled to room temperature, obtain Chinese medicine powder B after sterilizing, standby；
Step 6, the secondary seed solution of cultivation being inoculated in equipped with in the 50L fermentation tank of 32L liquid fermentation medium, inoculum concentration is the 12% of fermentation medium volume；Inoculate after terminating by 1:0.70 ventilation, 200rpm, pH6.8, cultivate 22h, obtain fermentation liquid for 36 DEG C；Adding Chinese medicine powder B after the sterilizing of its quality 2% in fermentation liquid, at 1:0.65 ventilation, 200rpm, pH6.8,36 DEG C are continued fermentation 12h and obtain final fermentation liquid, standby；
In described liquid fermentation medium, the mass percentage content of each composition is: glucose 2.0%, starch 1.8%, soybean cake powder 1.5%, peptone 1.5%, ammonium sulfate 1.3%；Calcium carbonate 0.03%, dipotassium hydrogen phosphate 0.03%, manganese phosphate 0.04%, calcium chloride 0.04%, surplus is water；
Step 7, by final for gained fermentation liquid transport pump to two effect vacuum concentration equipment in, one effect temperature 78 DEG C, vacuum 0.052MPa, two effects 52 DEG C, vacuum 0.062MPa, cocnentration factor is 3.5:1, concentration adds starch mix homogeneously in 15% ratio after terminating, it is delivered to be spray-dried top of tower by material with pump, it is atomized under 17000rpm by centrifugal type atomizer, carrying out and fluidized drying with the hot-air that inlet temperature is 150 DEG C, dried material reclaims after cyclone separator is drawn from splicing bucket, i.e. prepares bacillus subtilis Chinese medicine feed additive.
Prepared bacillus subtilis Chinese medicine feed additive water content is less than 6%, and viable count preserves 2 years at 1,800,000,000,000/g, additive, and bacillus subtilis therein still can preserve 95% bacterial activity.
Embodiment
4
The preparation method of a kind of bacillus subtilis Chinese medicine feed additive, comprises the following steps:
Step one, feeding bacillus subtilis is seeded on inclined-plane seed culture medium, cultivates 48h for 37 DEG C；In described inclined-plane seed culture medium, the mass percentage content of each composition is: glucose 1.3%, peptone 1.8%, Carnis Bovis seu Bubali cream 1.3%, yeast extract 1.9%, sodium chloride 0.5%, agar 1.8%, and surplus is water；
Step 2, from cultivate fresh inclined-plane picking 4 ring lawn be inoculated in the 250ml triangular flask equipped with 50ml fluid enlargement culture base, 36 DEG C, 190rpm shaking table shaken cultivation 22h, obtain seed liquor, standby；
In described fluid enlargement culture base, the mass percentage content of each composition is: glucose 1.4%, peptone 1.7%, Carnis Bovis seu Bubali cream 1.2%, yeast extract 1.3%, sodium chloride 0.5%, and surplus is water；
Step 3, take Radix Puerariae, Rhizoma Polygonati and meal wheat stone according to the ratio that mass ratio is 2:1:0.2, be placed in super micron mill and carry out micronizing, cross 77 mesh sieves, gained Chinese medicine powder is carried out high-temperature heat sterilization, is cooled to room temperature, obtain Chinese medicine powder A after sterilizing, standby；
Step 4, the seed liquor of above-mentioned cultivation being inoculated into equipped with in the 5L fermentation tank of 3L culture fluid, inoculum concentration is the 13% of culture fluid volume；After inoculation terminates, in 190rpm, 1:1 ventilation, pH 6.9, after cultivating 8h under the conditions of 35-37 DEG C, obtain bacterium solution；In bacterium solution, add Chinese medicine powder A after the sterilizing of its weight 6.7, after stirring, continue to be 230rpm, 1:1 ventilation, pH6.8, cultivate 11h under the conditions of 36 DEG C, obtain secondary seed solution, standby；
In described culture fluid, the mass percentage content of each composition is: glucose 1.3%, soybean cake powder 1.7%, peptone 2.3%, ammonium sulfate 1.08%, disodium hydrogen phosphate 0.13%, sodium dihydrogen phosphate 0.13%, magnesium sulfate 0.58%, calcium chloride 0.07%, and surplus is water；
Step 5, take Pericarpium Citri Reticulatae, Herba Houttuyniae and Fructus Crataegi for the ratio of 5:2:1 in mass ratio, be placed in super micron mill and carry out micronizing, cross 77 mesh sieves, gained Chinese medicine powder is carried out high-temperature heat sterilization, is cooled to room temperature, obtain Chinese medicine powder B after sterilizing, standby；
Step 6, the secondary seed solution of cultivation being inoculated in equipped with in the 50L fermentation tank of 33L liquid fermentation medium, inoculum concentration is the 12% of fermentation medium volume；Inoculation is cultivated 23h by 1:0.68 ventilation, 210rpm, pH6.7,35-37 DEG C, is obtained fermentation liquid after terminating；Adding Chinese medicine powder B after the sterilizing of its quality 2% in fermentation liquid, in 1:0.69 ventilation, 210rpm, pH6.8,35-37 DEG C is continued fermentation 12h and obtains final fermentation liquid, standby；
In described liquid fermentation medium, the mass percentage content of each composition is: glucose 2.4%, starch 1.8%, soybean cake powder 1.7%, peptone 1.7%, ammonium sulfate 1.8%；Calcium carbonate 0.03%, dipotassium hydrogen phosphate 0.04%, manganese phosphate 0.04%, calcium chloride 0.04%, surplus is water；
Step 7, by final for gained fermentation liquid transport pump to two effect vacuum concentration equipment in, one effect temperature 77 DEG C, vacuum 0.053MPa, two effects 53 DEG C, vacuum 0.062MPa, cocnentration factor is 3.7:1, concentration adds starch mix homogeneously in 18% ratio after terminating, it is delivered to be spray-dried top of tower by material with pump, it is atomized under 17000rpm by centrifugal type atomizer, carrying out and fluidized drying with the hot-air that inlet temperature is 145 DEG C, dried material reclaims after cyclone separator is drawn from splicing bucket, i.e. prepares bacillus subtilis Chinese medicine feed additive.
Prepared bacillus subtilis Chinese medicine feed additive water content is less than 6%, and viable count preserves 2 years at 1,400,000,000,000/g, additive, and bacillus subtilis therein still can preserve 95% bacterial activity.
Relevant animal is tested
Use by the bacillus acidophilus's feed additive in the present invention, its effect is carried out related experiment checking.Select 300 weanling young sheep, it is divided into three groups, often group 100, it is respectively test group, matched group and blank group, three groups feed different bacillus acidophilus's feed additives, the feedstuff of the bacillus acidophilus's feed additive prepared by the interpolation present invention that test group feeds after wean, adds normal diet additive, without feed additive in the sheep feedstuff that blank group feeds in the feedstuff of control group fed.Blank group, test group and the young sheep of control group fed wean allow young sheep high-performance as far as possible grow in the identical environment of other conditions, wean in the growth course of 6 weeks after statistics wean the young average daily ingestion amount of sheep, average daily gain and average sick rate, and average sick rate is 6 weeks all sick number of times of one group of son sheep and the ratio of young sheep quantity.Through statistics, result is as shown in table 1:
Table 1:
From data above, the average daily ingestion amount of the young sheep of test group and average daily gain the most relatively matched group and blank organize height, average sick rate is significant lower, bacillus acidophilus's feed additive prepared by the present invention can substantially increase the weight of animals, improve immunity, effectively alleviate that inappetence, dyspepsia, efficiency of feed utilization be low, premunition decline, poor growth, the problem such as even dead, thus improve the growth performance of young sheep.Growth to animal, has obvious facilitation.
Heretofore described feeding bacillus subtilis is common bacterial strain, can be commercially available by market.
Claims (2)
1. the preparation method of a bacillus subtilis Chinese medicine feed additive, it is characterised in that: comprise the following steps:
Step one, feeding bacillus subtilis is seeded on inclined-plane seed culture medium, cultivates 48h for 35-37 DEG C；In described inclined-plane seed culture medium, the mass percentage content of each composition is: glucose 0.5-1.5%, peptone 0.2-2%, Carnis Bovis seu Bubali cream 0.5-1.5%, yeast extract 0.5-1.5%, sodium chloride 0.5%, agar 1.5-2%, and surplus is water；
Step 2, from cultivate fresh inclined-plane picking 2-4 ring lawn be inoculated in the 250ml triangular flask equipped with 50ml fluid enlargement culture base, 35-37 DEG C, 180-240rpm shaking table shaken cultivation 16-24h, obtain seed liquor, standby；
In described fluid enlargement culture base, the mass percentage content of each composition is: glucose 0.5-1.5%, peptone 0.2-2%, Carnis Bovis seu Bubali cream 0.5-1.5%, yeast extract 0.5-1.5%, sodium chloride 0.5%, and surplus is water；
Step 3, take Radix Puerariae, Rhizoma Polygonati and meal wheat stone according to the ratio that mass ratio is 2:1:0.2, be placed in super micron mill and carry out micronizing, cross 60-80 mesh sieve, gained Chinese medicine powder is carried out high-temperature heat sterilization, is cooled to room temperature, obtain Chinese medicine powder A after sterilizing, standby；
Step 4, the seed liquor of above-mentioned cultivation being inoculated into equipped with in the 5L fermentation tank of 3L culture fluid, inoculum concentration is the 5-15% of culture fluid volume；After inoculation terminates, in 150-250rpm, 1:1 ventilation, after cultivating 8h under the conditions of pH6.5-7.0,35-37 DEG C, obtain bacterium solution；In bacterium solution, add Chinese medicine powder after the sterilizing of its weight 6%-7%, after stirring, continue to be 150-250rpm, 1:1 ventilation, cultivate 8-12h under the conditions of pH6.5-7.0,35-37 DEG C, obtain secondary seed solution, standby；
In described culture fluid, the mass percentage content of each composition is: glucose 0.5-1.5%, soybean cake powder 0.5-2.0%, peptone 0.5-2.5%, ammonium sulfate 0.25-1.5%, disodium hydrogen phosphate 0.05-0.20%, sodium dihydrogen phosphate 0.05-0.20%, magnesium sulfate 0.05-0.5%, calcium chloride 0.01-0.10%, surplus is water；
Step 5, take Pericarpium Citri Reticulatae, Herba Houttuyniae and Fructus Crataegi for the ratio of 5:2:1 in mass ratio, be placed in super micron mill and carry out micronizing, cross 60-80 mesh sieve, gained Chinese medicine powder is carried out high-temperature heat sterilization, is cooled to room temperature, obtain Chinese medicine powder B after sterilizing, standby；
Step 6, the secondary seed solution of cultivation being inoculated in equipped with in the 50L fermentation tank of 30-35L liquid fermentation medium, inoculum concentration is the 5-15% of fermentation medium volume；Inoculation is cultivated 20-25h by 1:0.6-0.75 ventilation, 150-220rpm, pH6.5-7.0,35-37 DEG C, is obtained fermentation liquid after terminating；Adding Chinese medicine powder B after the sterilizing of its quality 2% in fermentation liquid, in 1:0.6-0.75 ventilation, 150-220rpm, pH6.5-7.0,35-37 DEG C is continued fermentation 10-15h and obtains final fermentation liquid, standby；
In described liquid fermentation medium, the mass percentage content of each composition is: glucose 1.0-3.0%, starch 0.5-2.0%, soybean cake powder 0.5-2.0%, peptone 0.2-2%, ammonium sulfate 0.25-1.5%；Calcium carbonate 0.01-0.05%, dipotassium hydrogen phosphate 0.01-0.05%, manganese phosphate 0.01-0.05%, calcium chloride 0.01-0.05%, surplus is water；
Step 7, by in final for gained fermentation liquid transport pump to two effect vacuum concentration equipment, one effect temperature 75-80 DEG C, vacuum 0.05-0.055MPa, two effects 50-55 DEG C, vacuum 0.058-0.065MPa, cocnentration factor is 3-4:1, concentration adds starch mix homogeneously in 10-20% ratio after terminating, it is delivered to be spray-dried top of tower by material with pump, it is atomized under 15000-18000rpm by centrifugal type atomizer, carry out and fluidized drying with the hot-air that inlet temperature is 140-155 DEG C, dried material reclaims after cyclone separator is drawn from splicing bucket, i.e. prepare bacillus subtilis Chinese medicine feed additive.
2. the bacillus subtilis Chinese medicine feed additive prepared by preparation method of as claimed in claim 1 bacillus subtilis Chinese medicine feed additive institute, it is characterised in that: water content is less than 6%, and viable count is 10000-18000 hundred million/g.
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| CN113773996A (en) * | 2021-09-24 | 2021-12-10 | 南京工业大学 | Preparation method and application of feeding bacillus subtilis |
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Application publication date: 20160921 |