CN1682599A - Microbial fodder additive and its preparing method and use - Google Patents
Microbial fodder additive and its preparing method and use Download PDFInfo
- Publication number
- CN1682599A CN1682599A CNA2004100311967A CN200410031196A CN1682599A CN 1682599 A CN1682599 A CN 1682599A CN A2004100311967 A CNA2004100311967 A CN A2004100311967A CN 200410031196 A CN200410031196 A CN 200410031196A CN 1682599 A CN1682599 A CN 1682599A
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- liquid
- dry powder
- fermentation
- lactobacillus
- cfu
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- 229940049547 paraxin Drugs 0.000 description 1
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Fodder In General (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to microbial fodder additive, and a kind of microbial fodder additive comprising beneficial microbes and their metabolites and its preparation process and use. The microbial fodder additive is the mixture of fermented liquid or dry powder obtained with Bacillus subtilis, Bacillus cereus, lactobacillus and Saccharomyices cerevisiae and through high density deep layer liquid fermentation. It may be prepared into different specific liquid or powder preparations. The microbial fodder additive has high live bacillus content, long product preserving period and less foreign bacteria contamination, and has the functions of raise fodder utilization, promote animal's growth, increase animal product yield and quality, etc.
Description
Technical field
The present invention relates to a kind of microorganism feed addictive, specifically relate to a kind of microorganism feed addictive of forming by multiple beneficial microorganism and its meta-bolites (as proteolytic enzyme, lipase, amylase, cellulase etc.) and its production and use.
Background technology
The use of some promotes growth health-care agents has promoted the fast development of livestock industry in fodder industry and the aquaculture, but has brought many potential to threaten for again simultaneously the development of human health and feed industry.Promotes growth health-care agents such as abuse of antibiotics in present feed and the livestock and poultry cultivation, chemical synthetic drug, hormone, beta-stimulants, heavy metal, tranquilizer, cause a series of problems and consequence: (1) causes the animal products Chinese traditional medicine residual serious, directly be detrimental to health, even generation " three cause ", and make the animal products of China and fishery products when entering the world market, face thick-and-thin, suffer enormous economic loss every year.(2) cause the animal products quality to descend, lack the market competitiveness and trust.Arsenic preparation such as widely used p-aminophenyl arsine (arsenic) acid in feed is classified as carcinogen by international cancer research institution (IARC, 1980) at present.In addition, at the medium-term and long-term tranquilizer etc. that uses of pig industry, cause drug residue.China Ministry of Health report (1998), annual newly-increased 2,000,000 cancer patientss of China, wherein 1,000,000 people die.This makes the human consumer produce " phagophobia " to animal products.(3) cause bacterial drug resistance enhancing, Resistant strain to increase, medication as a result is invalid, threatens human security, and scientific circles are referred to as " bacterium vengeance ".The expert of the World Health Organization thinks, by the various resistance germs that livestock produces, can pass to the people by food, and produces the disease that is difficult to healing, harm humans health.Simultaneously, the microbiotic that duomycin, terramycin, paraxin, olaquindox, sulfonamide, Maduramicin etc. are easily residual and toxicity is bigger and a large amount of uses of synthesising bacteria anti-reflecting medicine, cause drug residue to increase, the Resistant strain diffusion, the animal flora imbalance, the immunizing power of animal is suppressed, the secondary superinfection.(4) cause environmental destruction.Environment protection becomes the problem that the people of other countries more and more are concerned about just day by day.The pollution that the intensification Production of Livestock and Poultry causes has become the important factor of restriction Production of Livestock and Poultry.According to EEC report, European nitrous oxide (N
2O) and have 33% and 34% in the discharge of methane amount respectively from its feeding field.Nitrous oxide can form nitrogen peroxide under photochemical effect, again can with ozonization, can cause skin carcinoma.In the intensive culture, livestock and poultry are discharged a large amount of ammonia-state nitrogens and phosphorus etc., and ammonia is the very strong material of a kind of contaminative, influence the growth of plant by changing water and soil earth acid-basicity.In addition, monogastric animal will be taken in intravital 70% phosphorus and drain owing to can't utilize phytate phosphorus, and high copper agent of use or arsenic preparation in the feed also can be through animal excrements and polluted source and arable land finally enter human body through food chain.At present some and unsafe promotes growth health-care agent have the production and sales of the enterprise of tame veterinary drug more than 1000 in China, reach 30,000,000,000 yuan, and this not only causes the residual increase of animal products, has also polluted environment.
In view of the problems that exist in above-mentioned feed and the livestock and poultry cultivation, the use of antibiotic etc additive has been subjected to human consumer's generally opposition, in recent years, the human consumer pays attention to safety, health and the health of food especially, formed the common recognition of " mankind of the food → health of healthy animal → safety ", thus very harsh to the requirement of food quality.For this reason, the firm measure that countries in the world have taked forbidding or restriction to use to microbiotic separately, all forbade microbiotic from 2006 as European Union, The World Health Organization (WHO) has proposed " reducing the global principle that microbiotic uses in edible animal ", China starts " feed safety engineering ", and has forbidden a collection of toxicity and residual bigger medicine.Face the strong request of the human consumer of countries in the world to food " safety, healthy, nutrition " and back to nature, each country is just at active development green feed additive or green promotes growth health-care agent.Microorganism feed addictive or claim little ecological active bacteria formulation, probiotics, natural, nontoxic with it, have no side effect, have no drug resistance, non-environmental-pollution, and can promote growth of animal and weightening finish, enhancing body immunity function, reduce the animal dead rate, improve food conversion ratio and advantage such as animal rearing benefit that the extensive approval and the parent that are subjected to countries in the world scientist and animal rearing person look at.Using microbe fodder additives in feed or animal rearing; alternative microbiotic or subdue the consumption of antibiotic etc significantly; improve the quality of animal products and the international competitiveness of local flavor and enhancing animal products; help continuing and sound development of livestock industry; help the health of animal and human's class, help environment protection.Therefore, exploitation and production micro organism green fodder additives have important reality, ecology and economic implications.
Microorganism feed addictive, be referred to as probiotics (Probiotics) in the world, its general definition be meant contain viable bacteria and (or) dead bacterium, the bacterial preparation that comprises its component and product, per os or drop into via other mucosal route, be intended to improve the balance of mucomembranous surface microorganism or enzyme, perhaps stimulate specificity or nonspecific immunity mechanism (1994, more than 40 latest definition that country participates in the German international conference).
Lactobacillus, yeast and genus bacillus be have separately the biology characteristics and the probiotic bacterium class of function.Wherein, lactobacillus is a kind of microorganism of surviving in anaerobism or few oxygen environment liked, it is the dominant microflora in the normal flora in the animal digestive tract, " be throughout one's life the companion; be throughout one's life benefit " with animal, bringing into play antagonism pathogenic micro-organism (as intestinal bacteria and Salmonellas etc.), start special and non-specific immunity system (strengthening immunity and disease resistance), reducing cholesterol, in and enterotoxin, synthesize nutritive substance, produce digestive enzymes and regulate function such as raising poultry nutritive metabolism.Yeast is the oxygen consumption microorganism, and it can not only help animal digestion, can also the endogenous beneficial microorganism vigor in promoting digestion road, replenish vitamin B group, and its cell wall polysaccharides also has the stimulation animal immunizing power, improves the effect of disease resistance.Benefit is given birth to the type genus bacillus; belong to oxygen consumption type microorganism; they can be grown rapidly after entering digestive tube; growth; produce very strong proteolytic enzyme; lipase; amylase; cellulase isoreactivity material; to protein; fat; the digestion of nutritive substances such as carbohydrate; important regulation is being brought into play in absorption and utilization; can also produce vitamins B and vitamin K; by reducing redox potential in the digestive tube; and produce a small amount of antimicrobial substance; come harmful microorganism is played inhibition and killing action; simultaneously, its spore is to warm; press; acid; alkali; enzyme and some drugs; poor environments such as X ray have very strong tolerance, process at feed; good stability in storage and the digestive tube; need not the special protection processing; just can survive for a long time, therefore, can be widely used in pig; ox; domestic animals such as chicken; fowl and aquatic animal.
At present, some Feed Enterprise are all at the microorganism feed addictive of actively seeking and study new environmental protection.But this type of microorganism feed addictive on the market generally mostly is a kind of single bacteria preparation that makes that uses in lactobacillus, yeast and the genus bacillus, function singleness, viable bacteria content is low in the product, production technique and equipment are more backward, the living bacterial cells survival time is short, inferior quality, and living contaminants is serious, and result of use is undesirable, unstable.
Summary of the invention
The objective of the invention is to utilize and comprise lactobacillus, yeast and genus bacillus are at the interior reasonable compound compatibility of three classes, 4 strain beneficial functions microorganisms, adopt liquid submerged fermentation technology and a series of after-processing technology technology, overcome existing microorganism feed addictive function singleness, viable bacteria content is low in the product, the living bacterial cells survival time is short, the product living contaminants is serious, result of use is undesirable, the unsettled defective of effect, thus a kind of viable bacteria content height is provided, the product shelf time is long, living contaminants is few, can improve the microorganism feed addictive of feed nutrient and efficiency of feed utilization.
Another object of the present invention is to provide the preparation method of described microorganism feed addictive.
A further object of the present invention is to provide the purposes of described microorganism feed addictive.
The objective of the invention is to realize by the following technical solutions:
The invention provides a kind of microorganism feed addictive, its liquor type is cultivated for subtilis (Bacillus subtilis), bacillus cereus (Bacillus cereus), lactobacillus (Lactobacillus sp.) and S. cervisiae (Saccharomyces cerevisiae) being carried out liquid submerged fermentation respectively, and the fermented liquid of each single bacterium mixed and get, the viable count of subtilis+bacillus cereus in the blended fermented liquid+lactobacillus+S. cervisiae has three specifications, promptly 〉=100,000,000/ml, 〉=10 hundred million/ml and 〉=100 hundred million/ml.
The liquid submerged femrentation culturing method of the fermented liquid of described subtilis (Bacillus subtilis) and bacillus cereus (Bacillus cereus) comprises following step:
1) dull and stereotypedly cultivates rejuvenation: subtilis and cured shape genus bacillus bacterial classification are adopted method streak inoculation on the genus bacillus plate culture medium of streak inoculation respectively, cultivated 12～24 hours in 25～35 ℃, make subtilis and the rejuvenation of cured shape genus bacillus respectively, and form single bacterium colony; Described genus bacillus plate culture medium is the mixture by following weightmeasurement ratio (W/V) preparation: peptone 0.5～1%, glucose 0.5～2%, extractum carnis 0.5～1.5%, potassium primary phosphate 0.3～0.6%, agar 3～4%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, standby;
2) make shake-flask seed: choose single bacterium colony respectively subtilis of cultivating from step 1) and the cured shape genus bacillus bacterium colony, be inoculated in the genus bacillus liquid substratum, in 28～37 ℃ of cultivations 20～30 hours, obtain subtilis and cured shape genus bacillus shake-flask seed liquid; Described genus bacillus liquid substratum is for pressing following weightmeasurement ratio (W/V) liquid mixture prepared: peptone 0.5～1%, glucose 0.5～2%, extractum carnis 0.5～1.5%, potassium primary phosphate 0.3～0.6%, 3.08% manganese sulfate solution 0.5～1.5%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, standby;
3) subtilis shake-flask seed liquid that production primary seed solution: with step 2) obtains and cured shape genus bacillus shake-flask seed liquid are with the inoculum size of 2～5v%, be inoculated in respectively in the first order seed liquid substratum of producing used first class seed pot, cultivate 18～24h in 32～37 ℃, stirring velocity 150～200rpm, air flow is that 1～2vols/vol/min (is abbreviated as: vvm), obtain the primary seed solution of subtilis and bacillus cereus respectively; Described first order seed liquid culture medium prescription is for pressing following weightmeasurement ratio (W/V) liquid mixture prepared: 120 order bean cake powders, 0.5～2.0%, 120 order fish meal, 0.2～1.5%, 120 order Semen Maydis powders 0.5～1.0%, glucose 1.0～4.5%, NaCl 0.1～0.3%, NaOH 0.05～0.10%, Na
2HPO
40.1～0.5%, synergistic agent 0.05～0.2%, defoamer 0.05～0.1%, and pH=7.5～7.6 through 121 ℃ of sterilizations 30～45 minutes, are cooled to 32～37 ℃; Described synergistic agent is open in first to file 200410008993.3, for by MgSO
40.1～0.5g/L, CaCl
21.0～4.0g/L, ZnSO
40.2～1.5g/L, FeSO
40.1～0.8g/L, CuSO
40.0 1～0.05g/L, Co (NO
3)
20.02～0.06g/L, MnSO
40.3 the aqueous solution that～1.5g/L forms;
4) second order fermentation (produce fermentation): subtilis and cured shape genus bacillus first order seed that step 3) is obtained, be inoculated into 5～10% volume ratio respectively and be equipped with in the fermentor tank of producing fermentation culture, the prescription of fermentation culture and control condition are same as the first order seed liquid substratum of step 3), ferment after 16～24 hours, by microscopic examination, stop fermentation when having a large amount of gemma to form.
When industrial production, when the turnout of needs more extensive (fermentor tank more than 20 tons), comprise that also step 5) carries out three grade fermemtation:
5) three grade fermemtation: subtilis and cured shape genus bacillus secondary seed that step 4) is obtained; ratio with 5～10% volumes is inoculated in the fermentor tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and control condition; ferment after 16～24 hours; by microscopic examination; when having a large amount of gemma to form, shut down.
The cultural method of the fermented liquid of described lactobacillus (Lactobacillus sp.) comprises following step:
1) dull and stereotyped cultivation rejuvenation:,, make the lactobacillus rejuvenation, and form single bacterium colony in 33～38 ℃ of cultivations 24～48 hours with method streak inoculation on the lactobacillus plate culture medium of lactobacillus inclined-plane seed employing streak inoculation; Described lactobacillus plate culture medium is the mixture by following weightmeasurement ratio (W/V) preparation: soy peptone 0.5～1%, glucose 1～2%, yeast soak powder 0.3～0.7%, potassium primary phosphate 0.3～0.5%, extractum carnis 0.5～1.5%, Triammonium citrate 1.5～2.3%, Tween-80 0.5～1.4%, sodium acetate 0.2～0.6%, agar powder 2～2.5%, pH=5.5～5.8, sterilized 15 minutes for 121 ℃, standby;
2) make shake-flask seed: choose single bacterium colony in the lactobacillus bacterium colony with the step 1) cultivation, be inoculated in the lactobacillus liquid nutrient medium, the employing ordinary method in 35～40 ℃ of cultivations 24～48 hours, obtains the lactobacillus shake-flask seed on the triangular flask liquid nutrient medium; Described substratum is by following weightmeasurement ratio (W/V) liquid mixture prepared: soy peptone 0.5～1%, glucose 1～2%, yeast soak powder 0.3～0.7%, potassium primary phosphate 0.3～0.5%, extractum carnis 0.5～1.5%, Triammonium citrate 1.5～2.3%, Tween-80 0.5～1.4%, sodium acetate 0.2～0.6%, pH=5.5～5.8, sterilized 15 minutes for 121 ℃, standby;
3) the lactobacillus shake-flask seed that production primary seed solution: with step 2) obtains is with the inoculum size of 6～12v%, be inoculated in the first order seed liquid substratum of producing used first class seed pot, cultivate 17～60h in 35～38 ℃, stirring velocity 150～200rpm, ventilation in 1 hour is 5～10 minutes at interval, air flow is 1～2.5vols/vol/min (vvm), reaches 1,000,000,000 until the lactobacillus number, obtains the primary seed solution of lactobacillus; Described first order seed liquid culture medium prescription is for pressing following weightmeasurement ratio (W/V) liquid mixture prepared: 120 order bean cake powders, 0.5～2.0%, 120 order fish meal 0.5～1.0%, fish peptone 0.5～2%, lactose 0.2～1.5%, 120 order Semen Maydis powders 0.5～1.0%, glucose 1～2.5%, NaCl 0.1～0.3%, Na
2HPO
40.15～0.3%, synergistic agent 0.05～0.3%, defoamer 0.03～0.1%, and pH=5.5～5.8 through 121 ℃ of sterilizations 30～45 minutes, are cooled to 32～37 ℃; Described synergistic agent is open in first to file 200410008992.9, for by CaCl
22～4g/L, ZnSO
40.5～1.5g/L, FeSO
40.2～1.0g/L, CuSO
40.01～0.08g/L, Co (NO
3)
20.02～0.06g/L, MgSO
40.1 the aqueous solution that～0.5g/L forms;
4) second order fermentation (produce fermentation): the lactobacillus first order seed that step 3) is obtained, be inoculated in the fermentor tank that the production fermentation culture is housed with 5～10% volume ratios, the prescription of fermentation culture and control condition are same as the first order seed liquid substratum of step 3), ferment after 17～60 hours, when detecting the pH value and dropping to 5.0 left and right sides (at this moment, the result of test of many times shows that the lactobacillus amount reaches greater than 10
9The CFU/ml fermented liquid) stops fermentation.
When industrial production, when the turnout of needs more extensive (fermentor tank more than 20 tons), comprise that also step 5) carries out three grade fermemtation:
5) three grade fermemtation: the lactobacillus secondary seed that step 4) is obtained; ratio with 5～10% volumes is inoculated in the fermentor tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and control condition; ferment after 17～60 hours; detection reaches 1,000,000,000/ml until the lactobacillus number, shuts down.
The cultural method of the fermented liquid of described yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) comprises following step:
1) dull and stereotyped cultivation rejuvenation:,, make the yeast saccharomyces cerevisiae rejuvenation, and form single bacterium colony in 24～30 ℃ of cultivations 20～24 hours with method streak inoculation on the yeast saccharomyces cerevisiae plate culture medium of yeast saccharomyces cerevisiae inclined-plane seed employing streak inoculation; Described yeast saccharomyces cerevisiae plate culture medium is the mixture by following weightmeasurement ratio (W/V) preparation: soy peptone 0.2～0.7%, glucose 0.5～1.5%, yeast soak powder 0.1～0.5%, malt extract 0.2～1%, agar powder 2～3%, pH=5.8～6.0, sterilized 15 minutes for 121 ℃, standby;
2) make shake-flask seed: from the yeast saccharomyces cerevisiae culture plate that step 1) is cultivated, choose single bacterium colony, be inoculated in the triangular flask liquid medium,, obtain the yeast saccharomyces cerevisiae shake-flask seed liquid in 28～32 ℃ of cultivations 24～37 hours; Described nutrient solution is by following weightmeasurement ratio (W/V) liquid mixture prepared: soy peptone 0.2～0.7%, glucose 0.5～1.5%, yeast soak powder 0.1～0.5%, malt extract 0.2～1%, pH=5.8～6.0, sterilized 15 minutes for 121 ℃, standby;
3) the yeast saccharomyces cerevisiae shake-flask seed that production primary seed solution: with step 2) obtains is by the inoculum size of 2～12v%, be inoculated in the first order seed nutrient solution of first class seed pot, cultivated 24～37 hours in 28～32 ℃, stirring velocity 160～220rpm, air flow is 1.0～3.0vols/vol/min (vvm), obtains the primary seed solution of yeast saccharomyces cerevisiae; Described yeast saccharomyces cerevisiae first order seed nutrient solution is for pressing following weightmeasurement ratio (W/V) liquid mixture prepared: 120 order bean cake powders, 0.5～1.5%, 120 order fish meal, 0.2～0.8%, 120 order Semen Maydis powders 0.5～1.0%, glucose 0.3～1.0%, NaCl 0.1～0.3%, Na
2HPO
40.2～0.5%, synergistic agent 0.05～0.2%, defoamer 0.03～0.1%, and pH=5.8～6.0 through 121 ℃ of sterilizations 30～45 minutes, are cooled to 32～37 ℃; Described synergistic agent is open in first to file 200410008994.8, for by MgSO
40.05～0.3g/L, CaCl
22.5～4g/L, ZnSO
40.6～1.5g/L, FeSO
40.1～1.0g/L, MnSO
40.4～0.8g/L, CuSO
40.02 the aqueous solution that～0.08g/L forms;
4) second order fermentation (produce fermentation): the yeast saccharomyces cerevisiae first order seed that step 3) is obtained, be inoculated in the fermentor tank that the production fermentation culture is housed with 4～10% volume ratios, the prescription of fermentation culture and control condition are same as the first order seed nutrient solution of step 3), ferment after 24～60 hours, detection S. cervisiae number stops fermentation when reaching 1,000,000,000/ml.
When industrial production, when the turnout of needs more extensive (fermentor tank more than 20 tons), comprise that also step 5) carries out three grade fermemtation:
5) three grade fermemtation: the yeast saccharomyces cerevisiae secondary seed that step 4) is obtained; ratio with 5～10% volumes is inoculated in the fermentor tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and control condition; ferment after 24～60 hours; detection reaches 1,000,000,000/ml until the S. cervisiae number, shuts down.
Microbe additive liquor provided by the invention is that above-mentioned fermentation of bacillus liquid, lactobacillus fermentation liquid, yeast fermentation broth are cultivated used substratum and the culture condition of rejuvenation according to flat board separately, viable bacteria content is carried out conventional plate count to be detected, according to count results, utilize aqua sterilisa that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml or 10
10CFU/ml, formulated according to following volume ratio according to practical use then:
The universal proportioning of liquor type microbe additive is:
Fermentation of bacillus subtilis liquid 10～30v%; Bacillus cereus fermented liquid 10～30v%;
Lactobacillus fermentation liquid 30～50v%; Yeast saccharomyces cerevisiae fermented liquid 10～30v%.
The proportioning of liquor type microbe additive young animal tailored version is:
Fermentation of bacillus subtilis liquid 10～20v%; Bacillus cereus fermented liquid 10～20v%;
Lactobacillus fermentation liquid 40～60v%; Yeast saccharomyces cerevisiae fermented liquid 15～25v%.
The proportioning of above the average age for marriage animal specific type is in the liquor type microbe additive:
Fermentation of bacillus subtilis liquid 20～40v%; Bacillus cereus fermented liquid 15～30v%;
Lactobacillus fermentation liquid 20～40v%; Yeast saccharomyces cerevisiae fermented liquid 10～25v%.
The proportioning of liquor type microbe additive ruminating animal (cattle and sheep etc.) tailored version is:
Fermentation of bacillus subtilis liquid 15～30v%; Bacillus cereus fermented liquid 10～30v%;
Lactobacillus fermentation liquid 30～40v%; Yeast saccharomyces cerevisiae fermented liquid 15～35v%.
The proportioning of liquor type microbe additive granulation feed tailored version is:
Fermentation of bacillus subtilis liquid 30～40v%; Bacillus cereus fermented liquid 20～30v%;
Lactobacillus fermentation liquid 20～30v%; Yeast saccharomyces cerevisiae fermented liquid 10～20v%.
Microorganism feed addictive provided by the invention, its powder-type is for to make dry powder with the fermented liquid of above-mentioned subtilis, bacillus cereus, lactobacillus and S. cervisiae through different after-processing technologies, mixture in following ratio preparation, the viable count of subtilis+bacillus cereus in the dry powder+lactobacillus+S. cervisiae has three specifications, promptly 〉=100,000,000/ml, 〉=10 hundred million/ml and 〉=100 hundred million/ml.
The preparation method of described subtilis dry powder and bacillus cereus dry powder comprises following step:
With the fermented liquid of subtilis and the fermented liquid of bacillus cereus, be that 0.5～0.8: 1 mixed is even according to the weight ratio of fermented liquid and carrier respectively, be the wet-milling shape, then on fluidizing drying bed in 80～100 ℃ of crushed after being dried to more than 80 orders, obtain subtilis dry powder and bacillus cereus dry powder respectively; Described carrier (sorbent material) proportioning is: lightweight CaCO
325～50wt%, inferior powder 20～30wt%, Zein powder 15～20wt%;
Or the fermented liquid of the fermented liquid of subtilis and bacillus cereus by volume added the dextrin of fermentating liquid volume 0.3～0.8% and 3～10% light calcium carbonate with weight ratio respectively, after mixing, on spray-drier, press 100～130 ℃ of air intakes, 60～90 ℃ of air-out, 2kg is pressed in spray, flow 1000～1500ml/min carries out spraying drying, obtains subtilis spraying dry powder and bacillus cereus spraying dry powder.
The preparation method of described dry powder of lactic acid bacteria comprises following step:
1) fermented liquid with above-mentioned milk-acid bacteria is concentrated to 40～60% of original volume through hot low temperature (＜45 ℃) negative-pressure vacuum;
2) adding accounts for the protective material of the weight/volume percent 15～20% of concentrated solution, mixes the back in-25～-45 ℃ of lyophilizes, obtains the lactic acid bacteria freeze drying powder; Described protective material be skim-milk, white sugar (sucrose), maltodextrin, gelatin by 1: 2: 1: 1 mixed forms;
Or the concentrated solution of milk-acid bacteria added in the carrier that is pre-mixed, the weight ratio of concentrated solution and carrier is 0.4～0.9: 1, through 45～60 ℃ of dryings of fluidized-bed, is crushed to more than 80 orders then, obtains dry powder of lactic acid bacteria; Described carrier comprises: lightweight CaCO
330～40wt%, inferior powder 15～30wt%, Zein powder 18～20wt%.
The preparation method of described S. cervisiae dry powder comprises following step:
1) fermented liquid with above-mentioned S. cervisiae is concentrated to 40～60% of original volume through hot low temperature (＜45 ℃) negative-pressure vacuum, obtains the concentrated solution of S. cervisiae;
2) concentrated solution of S. cervisiae is added in the carrier that is pre-mixed, the weight ratio of concentrated solution and carrier is 0.6～0.9: 1, fluidised bed drying then, and 45～60 ℃ of drying temperatures obtain S. cervisiae dry powder; Described carrier comprises: lightweight CaCO
330～50wt%, inferior powder 20～30wt%, Zein powder 18～20wt%.
Microbe additive pulvis provided by the invention is that above-mentioned genus bacillus dry powder, lactobacillus dry powder are cultivated used substratum and the culture condition of rejuvenation according to flat board separately, viable bacteria content to dry powder carries out conventional plate count detection, according to count results, utilize carrier that viable bacteria content is diluted to 10 respectively
8CFU/g, 10
9CFU/g or 10
10CFU/g, described carrier proportioning is: lightweight CaCO
325～50wt%, inferior powder 20～30wt%, Zein powder 15～20wt% form than preparation according to following weight according to practical use then:
The proportioning that the powder-type microbe additive is universal:
Subtilis dry powder 10～30wt%; Bacillus cereus dry powder 10～30wt%;
Lactobacillus dry powder 30～50wt%; S. cervisiae dry powder 10～30wt%.
The proportioning of powder-type microbe additive young animal tailored version is:
Subtilis dry powder 10～20wt%; Bacillus cereus dry powder 10～20wt%;
Lactobacillus dry powder 40～60wt%; S. cervisiae dry powder 15～25wt%.
The proportioning of above the average age for marriage animal specific type is in the powder-type microbe additive:
Subtilis dry powder 20～40wt%; Bacillus cereus dry powder 15～30wt%;
Lactobacillus dry powder 20～40wt%; S. cervisiae dry powder 10～25wt%.
The proportioning of powder-type microbe additive ruminating animal (cattle and sheep etc.) tailored version is:
Subtilis dry powder 15～30wt%; Bacillus cereus dry powder 10～30wt%;
Lactobacillus dry powder 30～40wt%; S. cervisiae dry powder 15～35wt%.
The proportioning of powder-type microbe additive granulation feed tailored version is:
Subtilis dry powder 30～40wt%; Bacillus cereus dry powder 20～30wt%;
Lactobacillus dry powder 20～30wt%; S. cervisiae dry powder 10～20wt%.
The quality standard of mentioned microorganism fodder additives provided by the invention is as follows:
Liquor: viable bacteria summation 〉=10 of 4 kinds of microorganisms
8/ milliliter (100,000,000), 10
9/ milliliter (1,000,000,000) and 10
10/ milliliter (10,000,000,000) 3 kinds of models or specification, all pale brown look liquid, denseer sour fragrance; Acidic liquid, pH≤4.5.
Pulvis: viable bacteria summation 〉=100,000,000 of 4 kinds of microorganisms/gram, 1,000,000,000/gram and 10,000,000,000/3 kinds of models of gram or specification is yellowish white powder, moisture content≤9%, ash≤8%, arsenic≤0.5mg/kg, lead≤1.0mg/kg.
The invention provides a kind of purposes of described microorganism feed addictive.This additive is as the microbiotic substitute or produce enzyme probiotics (enzyme-added probiotics), eliminate drug residue, improve product quality such as meat, eggs and milk, can be used for preventing and treating the various animals diarrhoea of having loose bowels, improve breeding performonce fo animals (increasing meat, egg, milk yield), improve immunizing power, disease resistance and feed efficiency, reduction animal death rate, also can be used for making ensiling, yellow storage feed and biological fodder, cotton dregs (cake) and the dish dregs of rice (cake) detoxification purify mew environment (ammonia falls in deodorizing) and purify fishes and shrimps pond water quality etc.
Microorganism feed addictive provided by the invention (Yishengkang) mainly is made up of proteolytic enzyme, lipase, amylase, cellulolytic enzyme and the bacteriostatic activity material of safe and efficient useful sporeformer, milk-acid bacteria, yeast etc. and microorganisms, selected bacterial classification belongs to the microbe species of united States food and drug administration and the announcement of U.S. feed management association, meet the GRAS standard, can be widely used in livestock and poultry and the aquatic products ecologic breeding, be non-harmful green feed additive.The characteristics that microorganism feed addictive provided by the invention utilizes yeast and genus bacillus can consume dissolved oxygen in the gi tract food, for endogenous anaerobism beneficial microorganism such as lactic acid bacteria class, bifidus bacillus etc. provide anaerobic environment, anaerobic environment can suppress to like the growth and breeding of oxygen harmful microorganism such as intestinal bacteria and Salmonellas etc. simultaneously, thereby safeguard the balance of microbial population of animal intestinal tract and the health of animal, brought into play the function of three class probioticss and intestines endogenous probiotics simultaneously, finally played promotes growth, enhance productivity and efficiency of feed utilization and health-care effect.Compare with the technology of existing microbe additive, at aspects such as zymotechnique, aftertreatment technology, viable bacteria content and stable effect, the advantage of microorganism feed addictive provided by the invention is:
1) this product has no side effect, has no drug resistance, noresidue, cost is low, effect is remarkable;
2) adopt deep liquid high density fermentation method to replace solid fermentation method, the viable bacteria content height, the production efficiency height, the viable bacteria in 3 kinds of specification products of liquor can reach 10 respectively
8/ milliliter, 10
9/ milliliter or 10
10/ milliliter, the viable bacteria in 3 kinds of specification products of pulvis also can reach 10
8/ g, 10
9/ g or 10
10/ g, different size, different formulation have stronger practicality or market adaptability applicable to different customer groups;
3) in the production process of product of the present invention, the one, adopt deep liquid high density fermentation technology (sterilization thoroughly, condition controllability strong), avoided the living contaminants that produces in the extensive solid fermentation process, or the thoroughly and during the fermentation not assorted bacterium breeding of sterilization, cause harmful bacterium and object bacteria (probiotics) to be mixed together growth, problem such as product is impure; The 2nd, the present invention is in aftertreatment technology, to carrier or sorbent material earlier through fluidizing drying bed at 105 ℃, 1～2 minute dry sterilization is handled, kill some the harmful bacterium in the support material, make the object bacteria composition purity height of product, clear efficacy, stable can not influence the effect of product because of the existence of assorted bacterium;
4) microorganism feed addictive of the present invention is 4 kinds of probioticss, scientific combination, and the ratio collocation is proper rationally, and multiple functional, clear and definite, effect is strong;
5) added synergistic agent during the fermentation, made total amount of probiotics improve several times to tens times, and prolonged the shelf lives of product, improved the stable effect of product than currently available products;
6) present method low cost product of producing is suitable for being extensive use of in fodder production and herding, aquaculture, has remarkable economic efficiency, social benefit and ecological benefits.
Embodiment
The fermented liquid (A) of embodiment 1, cultivation subtilis
1) dull and stereotypedly cultivates rejuvenation: the preservation bacterial classification of subtilis is adopted method streak inoculation on the genus bacillus plate culture medium of streak inoculation, cultivated 18 hours in 30 ℃, make the subtilis rejuvenation, and form single bacterium colony; Described bacillus subtilis bacterium culture medium is the mixture in following ratio preparation: peptone 0.5% (W/V, glucose 0.5%, extractum carnis 0.5%, potassium primary phosphate 0.3%, agar 3% down together),, pH=7.5～7.6,121 ℃ sterilization 30 minutes, standby;
2) bottle (triangular flask) seed is shaken in making: select typical single bacterium colony from the subtilis bacterium colony that step 1) is cultivated, be inoculated in the genus bacillus liquid substratum, in 32 ℃ of cultivations 30 hours, obtain the subtilis shake-flask seed liquid; Described genus bacillus liquid substratum is in following ratio liquid mixture prepared: peptone 0.5%, glucose 2%, extractum carnis 0.5%, potassium primary phosphate 0.6%, 3.08% manganese sulfate solution 1.0%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, standby;
3) production primary seed solution: earlier according to formulated first order seed liquid substratum described later, pH=7.5～7.6, through 121 ℃, sterilized 45 minutes, be cooled to 37 ℃, then with step 2) the subtilis shake-flask seed liquid that obtains is with the inoculum size of 2v%, be inoculated in respectively and produce in the used first class seed pot, cultivate 24h, stirring velocity 200rpm in 37 ℃, air flow is 2vvm, obtains the primary seed solution of subtilis; Described first order seed liquid culture medium prescription is in following ratio liquid mixture prepared (W/V), 120 order bean cake powders 1.0%, 120 order fish meal 1.0%, 120 order Semen Maydis powders 0.5%, glucose 2.0%, NaCl 0.2%, NaOH 0.08%, Na
2HPO
40.3%, synergistic agent 0.1%, defoamer 0.08%; Described synergistic agent is open in first to file 200410008993.3, for by MgSO
40.1～0.5g/L, CaCl
21.0～4.0g/L, ZnSO
40.2～1.5g/L, FeSO
40.1～0.8g/L, CuSO
40.01～0.05g/L, Co (NO
3)
20.02～0.06g/L, MnSO
40.3 the aqueous solution that～1.5g/L forms;
4) second order fermentation (produce fermentation): the subtilis first order seed that step 3) is obtained; ratio with 10% volume is inoculated in the fermentor tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and control condition; ferment after 20 hours; by microscopic examination; when having a large amount of gemma to form, shut down, obtain the fermented liquid of subtilis.At last according to dull and stereotyped used substratum and the culture condition of rejuvenation of cultivating of subtilis, the viable bacteria content of fermentation of bacillus subtilis liquid is carried out conventional plate count detect, according to count results, utilize the sterilization tap water that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml and 10
10CFU/ml obtains the reserve liquid (A) of fermentation of bacillus subtilis liquid, treats aftertreatment.
The fermented liquid (A) of embodiment 2, cultivation subtilis
1) dull and stereotypedly cultivates rejuvenation: the preservation bacterial classification of subtilis is adopted method streak inoculation on the genus bacillus plate culture medium of streak inoculation, cultivated 18 hours in 30 ℃, make the subtilis rejuvenation, and form single bacterium colony; Described bacillus subtilis bacterium culture medium is the mixture in following ratio preparation: (% refers to percent weight in volume to peptone 0.5%, glucose 0.5%, extractum carnis 0.5%, potassium primary phosphate 0.3%, agar 3% down together),, pH=7.5～7.6,121 ℃ sterilization 30 minutes, standby;
2) bottle (triangular flask) seed is shaken in making: select typical single bacterium colony from the subtilis bacterium colony that step 1) is cultivated, be inoculated in the genus bacillus liquid substratum, in 32 ℃ of cultivations 30 hours, obtain the subtilis shake-flask seed liquid; Described genus bacillus liquid substratum is in following ratio liquid mixture prepared: peptone 0.5%, glucose 2%, extractum carnis 0.5%, potassium primary phosphate 0.6%, 3.08% manganese sulfate solution 1.0%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, standby;
3) production primary seed solution: earlier according to formulated first order seed liquid substratum described later, pH=7.5～7.6, through 121 ℃, sterilized 45 minutes, be cooled to 37 ℃, then with step 2) the subtilis shake-flask seed liquid that obtains is with the inoculum size of 2v%, be inoculated in respectively and produce in the used first class seed pot, cultivate 24h, stirring velocity 200rpm in 37 ℃, air flow is 2vvm, obtains the primary seed solution of subtilis; Described first order seed liquid culture medium prescription is in following ratio liquid mixture prepared (W/V), 120 order bean cake powders 1.0%, 120 order fish meal 1.0%, 120 order Semen Maydis powders 0.5%, glucose 2.0%, NaCl 0.2%, NaOH 0.08%, Na
2HPO
40.3%, synergistic agent 0.1%, defoamer 0.08%; Described synergistic agent is open in first to file 200410008993.3, for by MgSO
40.1～0.5g/L, CaCl
21.0～4.0g/L, ZnSO
40.2～1.5g/L, FeSO
40.1～0.8g/L, CuSO
40.01～0.05g/L, Co (NO
3)
20.02～0.06g/L, MnSO
40.3 the aqueous solution that～1.5g/L forms;
4) second order fermentation (produce fermentation): the subtilis first order seed that step 3) is obtained; ratio with 10% volume is inoculated in the fermentor tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and control condition; ferment after 16 hours; shut down, obtain the secondary seed of subtilis.
5) three grade fermemtation: the subtilis secondary seed that step 4) is obtained; ratio with 5% volume is inoculated in the three grade fermemtation jar that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and control condition; ferment after 20 hours; by microscopic examination; when having a large amount of gemma to form, shut down, obtain fermentation of bacillus subtilis liquid.At last according to dull and stereotyped used substratum and the culture condition of rejuvenation of cultivating of subtilis, the viable bacteria content of fermentation of bacillus subtilis liquid is carried out conventional plate count detect, according to count results, utilize the sterilization tap water that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml and 10
10CFU/ml obtains the reserve liquid (A) of fermentation of bacillus subtilis liquid, treats aftertreatment.
The fermented liquid (B) of embodiment 3, cultivation bacillus cereus
Microbe species is bacillus cereus (Bacillus cereus), substratum, condition, preparation method are with embodiment 1, obtain the bacillus cereus fermented liquid, at last according to dull and stereotyped used substratum and the culture condition of rejuvenation of cultivating of Bacillus cereus, viable bacteria content to the Bacillus cereus fermented liquid carries out conventional plate count detection, according to count results, utilize the sterilization tap water that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml and 10
10CFU/ml obtains the reserve liquid (B) of bacillus cereus fermented liquid, treats aftertreatment.
The fermented liquid (B) of embodiment 4, cultivation bacillus cereus
1) microbe species is bacillus cereus (Bacillus cereus), and substratum, condition, preparation method obtain the bacillus cereus secondary seed solution with embodiment 2.
2) three grade fermemtation: the bacillus cereus secondary seed that step 1) is obtained; ratio with 8% volume is inoculated in the three grade fermemtation jar of fermentation culture; the prescription of fermentation culture and control condition are with step 2); ferment after 22 hours; by microscopic examination; when having a large amount of gemma to form; shut down; obtain the bacillus cereus fermented liquid; at last according to dull and stereotyped used substratum and the culture condition of rejuvenation of cultivating of Bacillus cereus; viable bacteria content to the Bacillus cereus fermented liquid carries out conventional plate count detection, according to count results, utilizes the sterilization tap water that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml and 10
10CFU/ml obtains the reserve liquid (B) of bacillus cereus fermented liquid, treats aftertreatment.
Embodiment 5, secondary are cultivated lactobacillus (Lactobacillus sp.) fermented liquid (C)
1) dull and stereotypedly cultivates rejuvenation: the lactobacillus inclined-plane seed of preservation is adopted method streak inoculation on the lactobacillus plate culture medium of streak inoculation, cultivated 24 hours in 33 ℃, make the lactobacillus rejuvenation, and form single bacterium colony; Described lactobacillus substratum is the mixture in following ratio preparation: soy peptone 1%, glucose 2%, yeast soak powder 0.7wt%, potassium primary phosphate 0.3%, extractum carnis 0.5%, Triammonium citrate 2.3%, Tween-80 1.4%, sodium acetate 0.2%, agar powder 2%, pH=5.5～5.8,121 ℃, sterilized 15 minutes, standby;
2) make shake-flask seed: select typical single bacterium colony in the lactobacillus bacterium colony with the step 1) cultivation, be inoculated in the lactobacillus liquid nutrient medium, the employing ordinary method in 35 ℃ of cultivations 30 hours, obtains the lactobacillus shake-flask seed on the triangular flask liquid nutrient medium; Described substratum is in following ratio liquid mixture prepared: soy peptone 1%, glucose 1%, yeast soak powder 0.7wt%, potassium primary phosphate 0.5%, extractum carnis 0.5%, Triammonium citrate 1.5%, Tween-80 0.5%, sodium acetate 0.6%, pH=5.5～5.8,115 ℃, sterilized 15 minutes, standby;
3) production primary seed solution:,, sterilized 45 minutes through 121 ℃ earlier according to formulated first order seed liquid substratum described later, pH=5.5～5.8, being cooled to 37 ℃, then with step 2) the lactobacillus shake-flask seed that obtains is with the inoculum size of 8v%, and be inoculated in and produce in the used first class seed pot, cultivate 30h in 35 ℃, stir fast 150rpm, ventilation in 1 hour is 5 minutes at interval, and air flow is 1.0vvm, reach 1,000,000,000 until the lactobacillus number, obtain lactobacillus and produce used primary seed solution; Described first order seed fermentation culture is in following ratio liquid mixture prepared (W/V): 120 order bean cake powders, 0.5,120 order fish meal 0.5%, fish peptone 0.5%, lactose 0.2%, 120 order Semen Maydis powder 0.5%, glucose 1%, NaCl 0.1%, Na
2HPO
40.15%, synergistic agent 0.05%, defoamer 0.03%, pH=5.5～5.8; Described synergistic agent is open in first to file 200410008992.9, for by CaCl
22～4g/L, ZnSO
40.5～1.5g/L, FeSO
40.2～1.0g/L, CuSO
40.01～0.08g/L, Co (NO
3)
20.02～0.06g/L, MgSO
40.1 the aqueous solution that～0.5g/L forms; Described substratum is same as step 2) in substratum;
4) second order fermentation (produce fermentation): the lactobacillus first order seed that step 3) is obtained, ratio with 10% volume is inoculated in the fermentor tank that the production fermentation culture is housed, the same step 3) of the prescription of fermentation culture and control condition, fermented 24 hours, obtain lactobacillus fermentation liquid, at last according to dull and stereotyped used substratum and the culture condition of rejuvenation of cultivating of lactobacillus, viable bacteria content to lactobacillus fermentation liquid carries out conventional plate count detection, according to count results, utilize the sterilization tap water that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml and 10
10CFU/ml obtains the reserve liquid (C) of lactobacillus fermentation liquid, treats aftertreatment.
Embodiment 6, three grade fermemtation lactobacillus liquid (C)
1) dull and stereotypedly cultivates rejuvenation: the lactobacillus inclined-plane seed of preservation is adopted method streak inoculation on the lactobacillus plate culture medium of streak inoculation, cultivated 24 hours in 33 ℃, make the lactobacillus rejuvenation, and form single bacterium colony; Described lactobacillus substratum is the mixture in following ratio preparation: soy peptone 1%, glucose 2%, yeast soak powder 0.7wt%, potassium primary phosphate 0.3%, extractum carnis 0.5%, Triammonium citrate 2.3%, Tween-80 1.4%, sodium acetate 0.2%, agar powder 2%, pH=5.5～5.8,121 ℃, sterilized 15 minutes, standby;
2) make shake-flask seed: select typical single bacterium colony in the lactobacillus bacterium colony with the step 1) cultivation, be inoculated in the lactobacillus liquid nutrient medium, the employing ordinary method in 35 ℃ of cultivations 30 hours, obtains the lactobacillus shake-flask seed on the triangular flask liquid nutrient medium; Described substratum is in following ratio liquid mixture prepared: soy peptone 1%, glucose 1%, yeast soak powder 0.7wt%, potassium primary phosphate 0.5%, extractum carnis 0.5%, Triammonium citrate 1.5%, Tween-80 0.5%, sodium acetate 0.6%, pH=5.5～5.8,115 ℃, sterilized 15 minutes, standby;
3) production primary seed solution:,, sterilized 45 minutes through 121 ℃ earlier according to formulated first order seed liquid substratum described later, pH=5.5～5.8, being cooled to 37 ℃, then with step 2) the lactobacillus shake-flask seed that obtains is with the inoculum size of 8v%, and be inoculated in and produce in the used first class seed pot, cultivate 30h in 35 ℃, stir fast 150rpm, ventilation in 1 hour is 5 minutes at interval, and air flow is 1.0vvm, reach 1,000,000,000 until the lactobacillus number, obtain lactobacillus and produce used primary seed solution; Described first order seed fermentation culture is in following ratio liquid mixture prepared (W/V): 120 order bean cake powders, 0.5,120 order fish meal 0.5%, fish peptone 0.5%, lactose 0.2%, 120 order Semen Maydis powder 0.5%, glucose 1%, NaCl 0.1%, Na
2HPO
40.1 5%, synergistic agent 0.05%, defoamer 0.03%, pH=5.5～5.8; Described synergistic agent is open in first to file 200410008992.9, for by CaCl
22～4g/L, ZnSO
40.5～1.5g/L, FeSO
40.2～1.0g/L, CuSO
40.01～0.08g/L, Co (NO
3)
20.02～0.06g/L, MgSO
40.1 the aqueous solution that～0.5g/L forms; Described substratum is same as step 2) in substratum;
4) second order fermentation (produce fermentation): the lactobacillus first order seed that step 3) is obtained, ratio with 10% volume is inoculated in the fermentor tank that the production fermentation culture is housed, the same step 3) of the prescription of fermentation culture and control condition, ferment after 30 hours, when detecting the pH value and dropping to 5.0 left and right sides (at this moment, the result of test of many times shows that the lactobacillus amount reaches greater than 10
9The CFU/ml fermented liquid); shut down; obtain the fermented liquid of lactobacillus; at last according to dull and stereotyped used substratum and the culture condition of rejuvenation of cultivating of lactobacillus; viable bacteria content to lactobacillus fermentation liquid carries out conventional plate count detection; according to count results, utilize the sterilization tap water that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml and 10
10CFU/ml obtains the reserve liquid (C) of lactobacillus fermentation liquid, treats aftertreatment.
Embodiment 7, second order fermentation are cultivated yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) fermented liquid (D)
1) dull and stereotypedly cultivates rejuvenation: the yeast saccharomyces cerevisiae inclined-plane seed of preservation is adopted method streak inoculation on the yeast saccharomyces cerevisiae plate culture medium of streak inoculation, cultivated 20～24 hours in 24～30 ℃, make the yeast saccharomyces cerevisiae rejuvenation, and form single bacterium colony; The mixture of described substratum for preparing by following ratio: soy peptone 0.5%, glucose 0.8%, yeast soak powder 0.3%, malt extract 0.7%, agar powder 3%, and sterilized 15 minutes in pH=5.8～6.0,121 ℃, standby;
2) make shake-flask seed: from the yeast saccharomyces cerevisiae culture plate that step 1) is cultivated, select typical single bacterium colony, be inoculated in the triangular flask liquid medium,, obtain the yeast saccharomyces cerevisiae shake-flask seed liquid in 28～32 ℃ of cultivations 24～37 hours; Described shake-flask seed nutrient solution is in following ratio liquid mixture prepared: soy peptone 0.5%, glucose 1.0%, yeast soak powder 0.4%, malt extract 0.8%, and sterilized 15 minutes in pH=5.8～6.0,121 ℃, standby;
3) production primary seed solution: earlier according to yeast saccharomyces cerevisiae culture medium prescription preparation first order seed liquid substratum described later, through 121 ℃, sterilized 45 minutes, pH=5.8～6.0 are cooled to 35 ℃, then with step 2) the yeast saccharomyces cerevisiae shake-flask seed that obtains is inoculated in the first class seed pot in the ratio of 10v%, cultivated 35 hours in 35 ℃, stirring velocity 180rpm, air flow is 1.5VVM, obtains yeast saccharomyces cerevisiae and produces used primary seed solution; Described yeast saccharomyces cerevisiae one-level kind nutrient solution is in following ratio liquid mixture prepared: 120 order bean cake powders 0.9%, 120 order fish meal 0.8%, 120 order Semen Maydis powders 0.9%, glucose 0.8%, NaCl 0.2%, Na
2HPO
40.5%, synergistic agent 0.1%, defoamer 0.1%; Described synergistic agent is open in first to file 200410008994.8, for by MgSO
40.05～0.3g/L, CaCl
22.5～4g/L, ZnSO
40.6～1.5g/L, FeSO
40.1～1.0g/L, MnSO
40.4～0.8g/L, CuSO
40.02 the aqueous solution that～0.08g/L forms.
4) second order fermentation (produce fermentation): the yeast saccharomyces cerevisiae first order seed that step 3) is obtained; ratio with 5% volume is inoculated in the fermentor tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and control condition; ferment after 36 hours; detection reaches 1,000,000,000/ml until the S. cervisiae number; shut down; obtain the fermented liquid of S. cervisiae; at last according to dull and stereotyped used substratum and the culture condition of rejuvenation of cultivating of yeast; viable bacteria content to yeast fermentation broth carries out conventional plate count detection; according to count results, utilize the sterilization tap water that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml and 10
10CFU/ml obtains the reserve liquid (D) of yeast fermentation broth, treats aftertreatment.
Embodiment 8, three grade fermemtation are cultivated yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) fermented liquid (D)
1) dull and stereotypedly cultivates rejuvenation: the yeast saccharomyces cerevisiae inclined-plane seed of preservation is adopted method streak inoculation on the yeast saccharomyces cerevisiae plate culture medium of streak inoculation, cultivated 20～24 hours in 24～30 ℃, make the yeast saccharomyces cerevisiae rejuvenation, and form single bacterium colony; The mixture of described substratum for preparing by following ratio: soy peptone 0.5%, glucose 0.8%, yeast soak powder 0.3%, malt extract 0.7%, agar powder 3%, and sterilized 15 minutes in pH=5.8～6.0,121 ℃, standby;
2) make shake-flask seed: from the yeast saccharomyces cerevisiae culture plate that step 1) is cultivated, select typical single bacterium colony, be inoculated in the triangular flask liquid medium,, obtain the yeast saccharomyces cerevisiae shake-flask seed liquid in 28～32 ℃ of cultivations 24～37 hours; Described shake-flask seed nutrient solution is in following ratio liquid mixture prepared: soy peptone 0.5%, glucose 1.0%, yeast soak powder 0.4%, malt extract 0.8%, and sterilized 15 minutes in pH=5.8～6.0,121 ℃, standby;
3) production primary seed solution: earlier according to yeast saccharomyces cerevisiae culture medium prescription preparation first order seed liquid substratum described later, through 121 ℃, sterilized 45 minutes, pH=5.8～6.0 are cooled to 32～37 ℃, then with step 2) the yeast saccharomyces cerevisiae shake-flask seed that obtains is by the inoculum size of 8v%, be inoculated in the first class seed pot,, stir fast 220rpm in 32 ℃ of cultivations 35 hours, air flow is 2.5VVM, obtains the yeast saccharomyces cerevisiae primary seed solution; Described yeast saccharomyces cerevisiae one-level kind nutrient solution is in following ratio liquid mixture prepared: 120 order bean cake powders 0.9%, 120 order fish meal 0.8%, 120 order Semen Maydis powders 0.9%, glucose 0.8%, NaCl 0.2%, Na
2HPO
40.5%, synergistic agent 0.1%, defoamer 0.03%; Described synergistic agent is open in first to file 200410008994.8, for by MgSO
40.05～0.3g/L, CaCl
22.5～4g/L, ZnSO
40.6～1.5g/L, FeSO
40.1～1.0g/L, MnSO
40.4～0.8g/L, CuSO
40.02 the aqueous solution that～0.08g/L forms.
4) second order fermentation (produce fermentation): the yeast saccharomyces cerevisiae first order seed that step 3) is obtained, ratio with 7% volume is inoculated in the fermentor tank that the production fermentation culture is housed, the same step 3) of the prescription of fermentation culture and control condition, fermenting obtains the secondary seed of S. cervisiae after 30 hours.
5) three grade fermemtation: the yeast saccharomyces cerevisiae secondary seed that step 4) is obtained; ratio with 5% volume is inoculated in the three grade fermemtation jar that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and control condition; ferment after 36 hours; detection reaches 1,000,000,000/ml until the S. cervisiae number; shut down; obtain fermentation by saccharomyces cerevisiae liquid; at last according to dull and stereotyped used substratum and the culture condition of rejuvenation of cultivating of yeast; viable bacteria content to yeast fermentation broth carries out conventional plate count detection; according to count results, utilize the sterilization tap water that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml and 10
10CFU/ml obtains the reserve liquid (D) of yeast fermentation broth.
Liquor benefit-1～the benefit-22 of embodiment 9～30, preparation microorganism feed addictive of the present invention
With the fermented liquid (D) of the S. cervisiae of the fermented liquid (C) of the lactobacillus of the fermented liquid (B) of the bacillus cereus of the fermented liquid (A) of the subtilis of embodiment 1～2, embodiment 3～4, embodiment 5～6 and embodiment 7～8 in the listed ratio preparation mixed solution of table 1, obtain the liquor (Yishengkang) of microorganism feed addictive of the present invention, i.e. benefit-1～benefit-22.
The liquor of table 1, microorganism feed addictive of the present invention
| Somatotype | Numbering | Embodiment number | The fermented liquid title | |||
| Subtilis A (v%) | Cured shape genus bacillus B (v%) | Lactobacillus C (v%) | Yeast saccharomyces cerevisiae D (v%) | |||
| Universal | Benefit-1 benefit-2 benefit-3 benefit-4 benefit-5 benefit-6 benefit-7 benefit-8 benefit-9 benefit-10 | ????9 | ????10 | ????30 | ????30 | ????30 |
| ????10 | ????30 | ????20 | ????35 | ????15 | ||
| ????11 | ????20 | ????10 | ????50 | ????20 | ||
| ????12 | ????30 | ????10 | ????40 | ????20 | ||
| ????13 | ????25 | ????25 | ????30 | ????20 | ||
| ????14 | ????10 | ????30 | ????35 | ????25 | ||
| ????15 | ????25 | ????25 | ????40 | ????10 | ||
| ????16 | ????30 | ????20 | ????30 | ????20 | ||
| ????17 | ????15 | ????15 | ????50 | ????20 | ||
| ????18 | ????30 | ????20 | ????40 | ????10 | ||
| The young animal tailored version | Benefit-11 | ????19 | ????10 | ????10 | ????60 | ????20 |
| Benefit-12 | ????20 | ????15 | ????20 | ????50 | ????15 | |
| Benefit-13 | ????21 | ????20 | ????15 | ????40 | ????25 | |
| In above the average age for marriage animal specific type | Benefit-14 benefit-15 benefits-16 | ????22 | ????20 | ????15 | ????40 | ????25 |
| ????23 | ????30 | ????25 | ????30 | ????15 | ||
| ????24 | ????40 | ????30 | ????20 | ????10 | ||
| The ruminating animal tailored version | Benefit-17 benefit-18 benefits-19 | ????25 | ????15 | ????30 | ????40 | ????15 |
| ????26 | ????30 | ????20 | ????30 | ????20 | ||
| ????27 | ????20 | ????10 | ????35 | ????35 | ||
| Granulation feed tailored version | Benefit-20 benefit-21 benefits-22 | ????28 | ????30 | ????30 | ????25 | ????15 |
| ????29 | ????35 | ????25 | ????30 | ????10 | ||
| ????30 | ????40 | ????20 | ????20 | ????20 | ||
Annotate: " benefit " is meant the code name of microbe additive name of product of the present invention " Yishengkang ".
Embodiment 31, preparation subtilis dry powder (E)
The fermentation of bacillus subtilis liquid that embodiment 1 or 2 is obtained is added in the carrier that is pre-mixed and mixes, and described carrier is lightweight CaCO
350wt%, inferior powder 20wt%, Zein powder 18% are formed, and the weight ratio of fermented liquid and carrier is 0.5: 1, then 45 ℃ of dryings;
Be crushed to then more than 80 orders, obtain subtilis dry powder respectively, at last according to dull and stereotyped used substratum and the culture condition of rejuvenation of cultivating of genus bacillus, viable bacteria content to dry powder carries out conventional plate count detection, according to count results, utilize the carrier of present embodiment that viable bacteria content is diluted to 10 respectively
8CFU/g, 10
9CFU/g and 10
10CFU/g obtains subtilis dry powder (E).
Embodiment 32, preparation subtilis dry powder (F)
The fermentation of bacillus subtilis liquid that embodiment 1 or 2 is obtained adds 0.5% (W/V) dextrin and 7% (W/V) light calcium carbonate, after mixing, on spray-drier, press 115 ℃ of air intakes, 80 ℃ of air-out, 2kg, flow 1000ml/min, spraying drying are pressed in spray, according to carrier and the quality control method of embodiment 31, viable bacteria content is diluted to 10 respectively then
8CFU/g, 10
9CFU/g and 10
10CFU/g obtains subtilis dry powder (F).
Embodiment 33, preparation bacillus cereus dry powder (G)
The bacillus cereus fermented liquid that embodiment 3 or 4 is obtained is added in the carrier that is pre-mixed, and described carrier is lightweight CaCO
345wt%, inferior powder 30wt%, Zein powder 20% are formed, and the weight ratio of fermented liquid and carrier is 0.8: 1, then 45 ℃ of dryings;
Be crushed to then more than 80 orders, obtain bacillus cereus dry powder respectively, at last according to dull and stereotyped used substratum and the culture condition of rejuvenation of cultivating of genus bacillus, viable bacteria content to dry powder carries out conventional plate count detection, according to count results, utilize the carrier of present embodiment that viable bacteria content is diluted to 10 respectively
8CFU/g, 10
9CFU/g and 10
10CFU/g, the bacillus cereus dry powder (G) that obtains.
Embodiment 34, preparation bacillus cereus dry powder (H)
The bacillus cereus fermented liquid that embodiment 3 or 4 is obtained volume ratio by weight adds 0.5% dextrin and 3% light calcium carbonate, after mixing, on spray-drier, press 100 ℃ of air intakes, 60 ℃ of air-out, 2kg is pressed in spray, and flow 1100ml/min carries out spraying drying, according to carrier and the quality control method of embodiment 33, viable bacteria content is diluted to 10 respectively then
8CFU/g, 10
9CFU/g and 10
10CFU/g obtains bacillus cereus dry powder (H).
Embodiment 35, preparation dry powder of lactic acid bacteria (I)
The lactobacillus fermentation liquid that embodiment 5 or 6 is obtained is concentrated to 40% of original volume through hot low temperature (＜45 ℃) negative-pressure vacuum, obtains concentrated solution.
Add then by skim-milk, white sugar (sucrose), maltodextrin, gelatin by 1: 2: 1: the protective material 15% of 1 mixed (accounting for the weight/volume percent of concentrated solution) mixes with the milk-acid bacteria concentrated solution;
To add protectant milk-acid bacteria concentrated solution in-45 ℃ of lyophilizes; at last, according to dull and stereotyped used substratum and the culture condition of rejuvenation of cultivating of lactobacillus, the viable bacteria content of dry powder is carried out conventional plate count detect; according to count results, utilize carrier that viable bacteria content is diluted to 10 respectively
8CFU/g, 10
9CFU/g and 10
10CFU/g obtains lactic acid bacteria freeze drying powder (I); Described carrier comprises: lightweight CaCO
330wt%, inferior powder 15wt%, Zein powder 18wt%.
Embodiment 36, preparation dry powder of lactic acid bacteria (J)
The lactobacillus fermentation liquid that embodiment 5 or 6 is obtained is concentrated to 50% of original volume through hot low temperature (＜45 ℃) negative-pressure vacuum, obtains concentrated solution.
This concentrated solution is added in the carrier that is pre-mixed, and described carrier is included as lightweight CaCO
335wt%, inferior powder 15wt%, Zein powder 20%, the weight ratio of concentrated solution and carrier is 0.5: 1, fluidised bed drying then, 45 ℃ of drying temperatures obtain dry powder of lactic acid bacteria, be crushed to more than 80 orders, at last, according to dull and stereotyped used substratum and the culture condition of rejuvenation of cultivating of lactobacillus, the viable bacteria content of dry powder is carried out conventional plate count detect, according to count results, utilize carrier that viable bacteria content is diluted to 10 respectively
8CFU/g, 10
9CFU/g and 10
10CFU/g obtains dry powder of lactic acid bacteria (J).
Embodiment 37, preparation S. cervisiae dry powder (K)
The yeast saccharomyces cerevisiae fermented liquid that embodiment 7 or 8 is obtained is concentrated to 40% of original volume through hot low temperature (＜45 ℃) negative-pressure vacuum, obtains concentrated solution.
This concentrated solution is added in the carrier that is pre-mixed, and described carrier is lightweight CaCO
340wt%, inferior powder 20wt%, Zein powder 20%, the weight ratio of concentrated solution and carrier is 0.7: 1, fluidised bed drying then, 50 ℃ of drying temperatures obtain S. cervisiae dry powder (K).
Pulvis benefit-23～the benefit-33 of embodiment 38～47, preparation microorganism feed addictive of the present invention
With the S. cervisiae dry powder (K) of the dry powder of lactic acid bacteria (J) of the dry powder of lactic acid bacteria (I) of the bacillus cereus dry powder (H) of the bacillus cereus dry powder (G) of the subtilis dry powder (F) of the subtilis dry powder (E) of embodiment 31, embodiment 32, embodiment 33, embodiment 34, embodiment 35, embodiment 36 and embodiment 37 in the listed ratio preparating mixture of table 2, obtain the pulvis (Yishengkang) of microorganism feed addictive of the present invention, i.e. benefit-23～benefit-44.
The pulvis of table 2, microorganism feed addictive of the present invention
| Embodiment number | Somatotype | Numbering | The composition wt% of various dry powder | ||||||
| The withered grass powder E (%) that drains off | Withered grass spray powder F (%) | The cured shape powder G (%) that drains off | Cured shape spray powder H (%) | Bacterium lacticum lyophilized powder I (%) | The Bacterium lacticum powder J (%) that drains off | The yeast powder K (%) that drains off | |||
| ????38 | Universal | Benefit-23 | ????20 | ????-- | ????30 | ????-- | ????30 | ????-- | ????20 |
| ????39 | Benefit-24 | ????15 | ????-- | ????-- | ????20 | ????40 | ????-- | ????25 | |
| ????40 | Benefit-25 | ????-- | ????30 | ????10 | ????15 | ????-- | ????35 | ????10 | |
| ????41 | Benefit-26 | ????10 | ????-- | ????10 | ????-- | ????50 | ????-- | ????30 | |
| ????42 | Benefit-27 | ????20 | ????10 | ????-- | ????20 | ????-- | ????35 | ????15 | |
| ????43 | Benefit-28 | ????-- | ????30 | ????10 | ????-- | ????50 | ????-- | ????10 | |
| ????44 | Benefit-29 | ????30 | ????-- | ????-- | ????30 | ????-- | ????30 | ????10 | |
| ????45 | Benefit-30 | ????20 | ????10 | ????10 | ????-- | ????40 | ????-- | ????20 | |
| ????46 | Benefit-31 | ????15 | ????10 | ????10 | ????10 | ????-- | ????35 | ????20 | |
| ????47 | Benefit-32 | ????10 | ????10 | ????-- | ????30 | ????30 | ????-- | ????20 | |
| ????48 | The young animal special use | Benefit-33 | ????10 | ????-- | ????-- | ????10 | ????-- | ????60 | ????20 |
| ????49 | Benefit-34 | ????-- | ????15 | ????20 | ????-- | ????50 | ????-- | ????15 | |
| ????50 | Benefit-35 | ????20 | ????-- | ????-- | ????15 | ????40 | ????-- | ????25 | |
| ????48 | In above the average age for marriage animal specific | Benefit-36 | ????20 | ????-- | ????-- | ????15 | ????40 | ????-- | ????25 |
| ????49 | Benefit-37 | ????-- | ????30 | ????25 | ????-- | ????-- | ????30 | ????15 | |
| ????50 | Benefit-38 | ????40 | ????-- | ????-- | ????30 | ????20 | ????-- | ????10 | |
| ????48 | The ruminating animal special use | Benefit-39 | ????-- | ????15 | ????-- | ????30 | ????40 | ????-- | ????15 |
| ????49 | Benefit-40 | ????-- | ????30 | ????20 | ????-- | ????-- | ????30 | ????20 | |
| ????50 | Benefit-41 | ????20 | ????-- | ????10 | ????-- | ????-- | ????35 | ????35 | |
| ????48 | It is special-purpose to granulate | Benefit-42 | ????30 | ????-- | ????30 | ????-- | ????25 | ????-- | ????15 |
| ????49 | Benefit-43 | ????-- | ????35 | ????25 | ????-- | ????-- | ????30 | ????10 | |
| ????50 | Benefit-44 | ????40 | ????-- | ????-- | ????20 | ????20 | ????-- | ????20 | |
Annotate: " powder drains off " is meant the fluidised bed drying powder in the table; " spray powder " is meant spray-drying powder." benefit " is meant the code name of microbe additive name of product of the present invention " Yishengkang ".
Embodiment 51, microorganism feed addictive of the present invention (Yishengkang, viable bacteria content 10
9/ ml or g) to the influence of milk yield and mastitis incidence
180 3 tire milk cows with about 650 kilograms of body weight are divided into 5 groups, and 30 every group, every component is 3 repetitions, and each repeats 10, and the grouping situation is as follows:
The A group, the blank group is only raised the milk cow basal diet;
The B group is added the Yishengkang group, and except that the feeding cow basal diet, feed per ton adds 1kg benefit-1 microorganism feed addictive;
The C group is added the Yishengkang group, and except that the feeding cow basal diet, feed per ton adds 1kg benefit-5 microorganism feed addictive;
The D group is added the Yishengkang group, and except that the feeding cow basal diet, feed per ton adds 1kg benefit-7 microorganism feed addictive;
The E group is added the Yishengkang group, and except that the feeding cow basal diet, feed per ton adds 1kg benefit-18 microorganism feed addictive;
The F group is added the Yishengkang group, and except that the feeding cow basal diet, feed per ton adds 1kg benefit-40 microorganism feed addictive;
Continuous 30 days, and press above-mentioned grouping revision test 4 times, record each group its milk yield of each detection and mastitis incidence are analyzed then, list in table 3.
The milk cow basal diet, prescription (weight percent) corn 45%, wheat bran 10%, rapeseed cake 9%, cotton dregs 10.0%, dry beer vinasse 5.0%, soybean meal 18.0%, stone flour 1.0%, salt 1.0%, Preblend 1%.
Milk production of cow and mastitis incidence before and after table 3, the test
| Group/performance | Tested preceding 15 days | Tested back 15 days | ||
| Per day milk yield (kg) | Mastitis incidence (%) | Average milk yield (kg) | Mastitis incidence (%) | |
| A organizes (blank) | ????33.45 | ????5.3 | ????34.76 | ????5.7 |
| B group (benefit-1) | ????36.40 | ????1.2 | ????37.86 | ????1.1 |
| C group (benefit-5) | ????36.51 | ????1.5 | ????37.98 | ????1.3 |
| D group (benefit-7) | ????36.10 | ????1.6 | ????37.84 | ????1.0 |
| E group (benefit-18) | ????36.24 | ????1.5 | ????38.07 | ????1.1 |
| F group (benefit-40) | ????35.98 | ????1.4 | ????38.85 | ????1.2 |
Can find out from table 3, Yishengkang can significantly improve the per day milk yield of milk cow, and obviously reduce the mastitis incidence of milk cow, and adding many and early stage in later stage on the daily yielding, on breast rate developmental rate, later stage, this illustrated along with the Yishengkang prolongation of duration of service to have storage effect less than early stage.To sum up test shows that Yishengkang can significantly improve the per day milk yield of milk cow, and can reduce the mastitis incidence of milk cow, and major cause is because the Yishengkang microbe additive can improve due to the immunizing power of milk cow.
Embodiment 52, microorganism feed addictive of the present invention (Yishengkang, viable bacteria content 10
8/ ml or g) to the influence of laying hen production performance, egg product matter, pouity dwelling place environment
15000 of brown shell layers are matched in the later stage sea of laying eggs that shared age in days is identical, divide 5 groups, and one group 3000 of every buildings divide 3 repetitions, and each repeats 1000.The grouping situation is as follows:
The A group, blank group, the laying hens essential ration of feeding;
The B group is added the Yishengkang group, adds 1kg benefit-6 microorganism feed addictive in laying hens essential ration;
The C group is added the Yishengkang group, adds 1kg benefit-23 microorganism feed addictive in laying hens essential ration;
The D group is added the Yishengkang group, adds 1kg benefit-27 microorganism feed addictive in laying hens essential ration;
The E group is added the Yishengkang group, adds 1kg benefit-38 microorganism feed addictive in laying hens essential ration;
The laying hens essential ration prescription (weight percent) that uses is: corn 58.72%, dregs of beans 26.0%, wheat bran 2.0%, stone flour 8.44%, hydrogen calcium 1.5%, salt 0.34%, vegetables oil 2.0%, 1% Preblend 1.0%.
Preceding 7 days is preliminary trial period, and back 23 days is trial period, and presses above-mentioned grouping revision test 3 times, has measured NH in the house
3, H
2Indexs such as S concentration, broken rate of eggshell, protein mass (Hough unit), laying rate, egg size, death rate, ight soil state, eggshell color and luster.The result is as shown in table 4.
Table 4,
As can be seen from Table 4, the Yishengkang additive provided by the invention of feeding, every index of egg illustrates that all significantly better than control group this activated peptide as additive has played good promoter action in production performance that improves laying hen and egg product matter.Because of Yishengkang can be regulated the intestinal microflora balance, promote the secretory volume of microbial enzyme, improve digestive enzyme activity, and then plain absorption of raising feed nitrogen nutrition transforms and utilising efficiency, the content of ammonia and hydrogen sulfide has good economy and ecological benefits in the indirect reduction pouity dwelling place.
Embodiment 53～56, microorganism feed addictive of the present invention (Yishengkang) are to the influence of piglet, growing and fattening pigs, fryer and grass carp
Table 5,
| Embodiment number | Yishengkang | Test method | Experimental scale | Test-results |
| 53 | Benefit-4 benefit-11 benefit-13 benefit-33 benefit-34 benefits-35 all contain bacterium (1010/ g, ml) | With embodiment 52, the A group adds duomycin 100g/t contrast, B, C, D, E, F, the G group is added benefit-4,11,13,33,34,35 each 0.1kg/t, every group of 30 pigs, 3 repetitions. | 210 of piglets | Contrast with adding the antibiosis group, the indices that adds the Yishengkang group improves as follows: daily ingestion amount raising 9.3%～11.3% daily gain improves 10.6%～12.1% feed efficiency improvement, 12.8%～13.4% diarrhea rate and reduces by 53.3%～57.8% |
| 54 | A: contrast B: benefit-8 C: benefit-10 D: benefit-14 E: benefit-15 F: benefit-16 (108/ g, ml) | With embodiment 52, the A group adds duomycin 100g/t, sulfanilamide (SN) 100g/t contrast, B, C, D, E, the F group is added benefit-8,10,14,15,16 each 1.5kg/t, every group of 60 pigs, 6 repetitions. | Breed 360 of growing and fattening pigs (30kg-listing) | Each growth phase food consumption of fattening pig, day weight gain; Feed efficiency improves, diarrhea rate and death rate reduce, and every production performance is not less than adds microbiotic colony; The pig house stink reduces, and dressing percentage is similar or higher to interpolation microbiotic colony with lean ratio, and the pork yellowish pink is ruddy, and is Fresh ﹠ Tender in Texture. |
| 55 | Chicken is added: A: contrast B: benefit-2 C: benefit-3 D: benefit-9 E: benefit-12 F: benefit-32 (contains bacterium 108) middle chicken interpolation: benefit-24 | Big chicken is added: benefit-28 benefit-29 benefit-30 benefit-31 benefits-38 (contain bacterium 10 10) contrast adds terramycin 150g/t chicken and add among the 1.5kg/t chicken and add the big chicken of 150kg/t and add 20g/t | 24000 of fryer | Increase the deposition of chicken body internal protein and reduce abdomen fat rate (P＜0.05), obviously reduce duodenum, pH value (p＜0.05 of jejunum and ileum,), significantly suppress the E.Coli breeding in (minimizing) jejunum and the rectum, and can improve the content (p＜0.05) of lactobacillus in the enteron aisle, very help the health of chicken and reduce stink. |
| Benefit-25 benefit-26 benefit-36 benefits-37 (contain bacterium 10 9) | Feed granulating, every group of 4000 fryer, 4 repetitions, each repeats 1000, totally 24000 | |||
| 56 | A: blank B: benefit-20 C: benefit-21 D: benefit-22 E: benefit-42 F: benefit-43 G: benefit-44 (bacteria containing amounts 109/ g, ml) | Feed granulating, every group of 3000 grass carps, 3 repetitions, each repeats 1000, totally 21000 | 21000 finishing period grass carps | Feed coefficient improves 6% than control group, day weight gain improves 12.3% than control group, surviving rate improves 20.7%, and the reguarity of test group is good, and resistance against diseases is strong.Water quality control aspect, the depth of water is thrown in Yishengkang liquid 1kg for 2.8 meters, 3 mu.Ammonia-state nitrogen (NH as a result 3-N) in 24 hours, be reduced to the 0.2mg/L of test group by the 0.6mg/L of control group, reduce by 3 times, the white skin disease of tilapia reduces 61.3% simultaneously. |
As can be seen from Table 5, the Yishengkang additive provided by the invention of feeding, every index of piglet, the difference raising stage of breeding pig, broiler chicken and grass carp is all significantly better than control group (interpolation microbiotic), illustrate this green feed additive can effectively prevent and treat livestock and poultry and aquatic animal disease, significantly improve production performance (weightening finish, feed efficiency etc.), improve animal house environment and aquatic animal water quality, improve effects such as animal product quality, good economy and ecological benefits are arranged.
Claims (18)
1, a kind of microorganism feed addictive, it comprises fermented liquid or dry powder with subtilis, bacillus cereus, lactobacillus and S. cervisiae difference fermentation culture.
2, microorganism feed addictive as claimed in claim 1 is characterized in that, the fermented liquid of described subtilis and bacillus cereus is to be obtained by liquid submerged fermentation, comprises following step:
1) dull and stereotypedly cultivates rejuvenation: subtilis and cured shape genus bacillus bacterial classification are adopted method streak inoculation on the genus bacillus plate culture medium of streak inoculation respectively, cultivated 12～24 hours in 25～35 ℃, make subtilis and the rejuvenation of cured shape genus bacillus respectively, and form single bacterium colony; Described genus bacillus plate culture medium is the mixture by following weightmeasurement ratio preparation: peptone 0.5～1%, glucose 0.5～2%, extractum carnis 0.5～1.5%, potassium primary phosphate 0.3～0.6%, agar 3～4%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, standby;
2) make shake-flask seed: choose single bacterium colony respectively subtilis of cultivating from step 1) and the cured shape genus bacillus bacterium colony, be inoculated in the genus bacillus liquid substratum, in 28～37 ℃ of cultivations 20～30 hours, obtain subtilis and cured shape genus bacillus shake-flask seed liquid; Described genus bacillus liquid substratum is for pressing following weightmeasurement ratio liquid mixture prepared: peptone 0.5～1%, glucose 0.5～2%, extractum carnis 0.5～1.5%, potassium primary phosphate 0.3～0.6%, 3.08% manganese sulfate solution 0.5～1.5%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, standby;
3) subtilis shake-flask seed liquid that production primary seed solution: with step 2) obtains and cured shape genus bacillus shake-flask seed liquid are with the inoculum size of 2～5v%, be inoculated in respectively in the first order seed liquid substratum of producing used first class seed pot, cultivate 18～24h in 32～37 ℃, stirring velocity 150～200rpm, air flow is 1～2vols/vol/min, obtains the primary seed solution of subtilis and bacillus cereus respectively; Described first order seed liquid culture medium prescription is for pressing following weightmeasurement ratio liquid mixture prepared: 120 order bean cake powders, 0.5～2.0%, 120 order fish meal, 0.2～1.5%, 120 order Semen Maydis powders 0.5～1.0%, glucose 1.0～4.5%, NaCl 0.1～0.3%, NaOH 0.05～0.10%, Na
2HPO
40.1～0.5%, synergistic agent 0.05～0.2%, defoamer 0.05～0.1%, and pH=7.5～7.6 through 121 ℃ of sterilizations 30～45 minutes, are cooled to 32～37 ℃; Described synergistic agent is by MgSO
40.1～0.5g/L, CaCl
21.0～4.0g/L, ZnSO
40.2～1.5g/L, FeSO
40.1～0.8g/L, CuSO
40.01～0.05g/L, Co (NO
3)
20.02～0.06g/L, MnSO
40.3 the aqueous solution that～1.5g/L forms;
4) second order fermentation: subtilis and cured shape genus bacillus first order seed that step 3) is obtained, be inoculated into 5～10% volume ratio respectively and be equipped with in the fermentor tank of producing fermentation culture, the prescription of fermentation culture and control condition are same as the first order seed liquid substratum of step 3), ferment after 16～24 hours, by microscopic examination, stop fermentation when having a large amount of gemma to form.
3, microorganism feed addictive as claimed in claim 1 is characterized in that, the process for preparation of dry powder of described subtilis and bacillus cereus comprises following step:
With the fermented liquid of subtilis and the fermented liquid of bacillus cereus, weight ratio according to fermented liquid and carrier is 0.5～0.8 respectively: 1 mixes, then on fluidizing drying bed in 80～100 ℃ of crushed after being dried to more than 80 orders, obtain subtilis dry powder and bacillus cereus dry powder respectively; Described carrier proportioning is: lightweight CaCO
325～50wt%, inferior powder 20～30wt%, Zein powder 15～20wt%;
Or the fermented liquid of the fermented liquid of subtilis and bacillus cereus by volume added the dextrin of fermentating liquid volume 0.3～0.8% and 3～10% light calcium carbonate with weight ratio respectively, after mixing, on spray-drier, press 100～130 ℃ of air intakes, 60～90 ℃ of air-out, 2kg is pressed in spray, flow 1000～1500ml/min carries out spraying drying, obtains subtilis spraying dry powder and bacillus cereus spraying dry powder.
4, microorganism feed addictive as claimed in claim 1 is characterized in that, the fermented liquid of described lactobacillus is to be obtained by liquid submerged fermentation, comprises following step:
1) dull and stereotyped cultivation rejuvenation:,, make the lactobacillus rejuvenation, and form single bacterium colony in 33～38 ℃ of cultivations 24～48 hours with method streak inoculation on the lactobacillus plate culture medium of lactobacillus inclined-plane seed employing streak inoculation; Described lactobacillus plate culture medium is the mixture by following weightmeasurement ratio preparation: soy peptone 0.5～1%, glucose 1～2%, yeast soak powder 0.3～0.7%, potassium primary phosphate 0.3～0.5%, extractum carnis 0.5～1.5%, Triammonium citrate 1.5～2.3%, Tween-80 0.5～1.4%, sodium acetate 0.2～0.6%, agar powder 2～2.5%, pH=5.5～5.8, sterilized 15 minutes for 121 ℃, standby;
2) make shake-flask seed: choose single bacterium colony in the lactobacillus bacterium colony with the step 1) cultivation, be inoculated in the lactobacillus liquid nutrient medium, the employing ordinary method in 35～40 ℃ of cultivations 24～48 hours, obtains the lactobacillus shake-flask seed on the triangular flask liquid nutrient medium; Described substratum is by following weightmeasurement ratio liquid mixture prepared: soy peptone 0.5～1%, glucose 1～2%, yeast soak powder 0.3～0.7%, potassium primary phosphate 0.3～0.5%, extractum carnis 0.5～1.5%, Triammonium citrate 1.5～2.3%, Tween-80 0.5～1.4%, sodium acetate 0.2～0.6%, pH=5.5～5.8, sterilized 15 minutes for 121 ℃, standby;
3) the lactobacillus shake-flask seed that production primary seed solution: with step 2) obtains is with the inoculum size of 6～12v%, be inoculated in the first order seed liquid substratum of producing used first class seed pot, cultivate 17～60h in 35～38 ℃, stirring velocity 150～200rpm, ventilation in 1 hour is 5～10 minutes at interval, air flow is 1～2.5vols/vol/min, reaches 1,000,000,000 until the lactobacillus number, obtains the primary seed solution of lactobacillus; Described first order seed liquid culture medium prescription is for pressing following weightmeasurement ratio liquid mixture prepared: 120 order bean cake powders, 0.5～2.0%, 120 order fish meal 0.5～1.0%, fish peptone 0.5～2%, lactose 0.2～1.5%, 120 order Semen Maydis powders 0.5～1.0%, glucose 1～2.5%, NaCl 0.1～0.3%, Na
2HPO
40.15～0.3%, synergistic agent 0.05～0.3%, defoamer 0.03～0.1%, and pH=5.5～5.8 through 121 ℃ of sterilizations 30～45 minutes, are cooled to 32～37 ℃; Described synergistic agent is by CaCl
22～4g/L, ZnSO
40.5～1.5g/L, FeSO
40.2～1.0g/L, CuSO
40.01～0.08g/L, Co (NO
3)
20.02～0.06g/L, MgSO
40.1 the aqueous solution that～0.5g/L forms;
4) second order fermentation: the lactobacillus first order seed that step 3) is obtained, be inoculated in the fermentor tank that the production fermentation culture is housed with 5～10% volume ratios, the prescription of fermentation culture and control condition are same as the first order seed liquid substratum of step 3), ferment after 17～60 hours, detection pH value drops at 5.0 o'clock and stops fermentation.
5, microorganism feed addictive as claimed in claim 1 is characterized in that, the preparation method of described dry powder of lactic acid bacteria comprises following step:
1) fermented liquid of milk-acid bacteria is concentrated to 40～60% of original volume being lower than under 45 ℃, negative-pressure vacuum condition;
2) adding accounts for the protective material of the weight/volume percent 15～20% of concentrated solution, mixes the back in-25～-45 ℃ of lyophilizes, obtains the lactic acid bacteria freeze drying powder; Described protective material be skim-milk, white sugar, maltodextrin, gelatin by 1: 2: 1: 1 mixed forms;
Or the concentrated solution of milk-acid bacteria added in the carrier that is pre-mixed, the weight ratio of concentrated solution and carrier is 0.4～0.9: 1, through 45～60 ℃ of dryings of fluidized-bed, is crushed to more than 80 orders then, obtains dry powder of lactic acid bacteria; Described carrier comprises: lightweight CaCO
330～40wt%, inferior powder 15～30wt%, Zein powder 18～20wt%.
6, microorganism feed addictive as claimed in claim 1 is characterized in that, the fermented liquid of described yeast saccharomyces cerevisiae is to be obtained by liquid submerged fermentation, comprises following step:
1) dull and stereotyped cultivation rejuvenation:,, make the yeast saccharomyces cerevisiae rejuvenation, and form single bacterium colony in 24～30 ℃ of cultivations 20～24 hours with method streak inoculation on the yeast saccharomyces cerevisiae plate culture medium of yeast saccharomyces cerevisiae inclined-plane seed employing streak inoculation; Described yeast saccharomyces cerevisiae plate culture medium is the mixture by following weightmeasurement ratio preparation: soy peptone 0.2～0.7%, glucose 0.5～1.5%, yeast soak powder 0.1～0.5%, malt extract 0.2～1%, agar powder 2～3%, pH=5.8～6.0, sterilized 15 minutes for 121 ℃, standby;
2) make shake-flask seed: from the yeast saccharomyces cerevisiae culture plate that step 1) is cultivated, choose single bacterium colony, be inoculated in the triangular flask liquid medium,, obtain the yeast saccharomyces cerevisiae shake-flask seed liquid in 28～32 ℃ of cultivations 24～37 hours; Described nutrient solution is by following weightmeasurement ratio liquid mixture prepared: soy peptone 0.2～0.7%, glucose 0.5～1.5%, yeast soak powder 0.1～0.5%, malt extract 0.2～1%, and pH=5.8～6.0,121 ℃ sterilization 15 minutes is standby;
3) the yeast saccharomyces cerevisiae shake-flask seed that production primary seed solution: with step 2) obtains is by the inoculum size of 2～12v%, be inoculated in the first order seed nutrient solution of first class seed pot, cultivated 24～37 hours in 28～32 ℃, stirring velocity 160～220rpm, air flow is 1.0～3.0vols/vol/min, obtains the primary seed solution of yeast saccharomyces cerevisiae; Described yeast saccharomyces cerevisiae first order seed nutrient solution is for pressing following weightmeasurement ratio liquid mixture prepared: 120 order bean cake powders, 0.5～1.5%, 120 order fish meal, 0.2～0.8%, 120 order Semen Maydis powders 0.5～1.0%, glucose 0.3～1.0%, NaCl 0.1～0.3%, Na
2HPO
40.2～0.5%, synergistic agent 0.05～0.2%, defoamer 0.03～0.1%, and pH=5.8～6.0 through 121 ℃ of sterilizations 30～45 minutes, are cooled to 32～37 ℃; Described synergistic agent is by MgSO
40.05～0.3g/L, CaCl
22.5～4g/L, ZnSO
40.6～1.5g/L, FeSO
40.1～1.0g/L, MnSO
40.4～0.8g/L, CuSO
40.02 the aqueous solution that～0.08g/L forms;
4) second order fermentation: the yeast saccharomyces cerevisiae first order seed that step 3) is obtained, be inoculated in the fermentor tank that the production fermentation culture is housed with 4～10% volume ratios, the prescription of fermentation culture and control condition are same as the first order seed nutrient solution of step 3), ferment after 24～60 hours, detection S. cervisiae number stops fermentation when reaching 1,000,000,000/ml.
7, microorganism feed addictive as claimed in claim 1 is characterized in that, the preparation method of described S. cervisiae dry powder comprises following step:
1) fermented liquid of S. cervisiae is concentrated to 40～60% of original volume being lower than under 45 ℃, the condition of negative-pressure vacuum, obtains the concentrated solution of S. cervisiae;
2) concentrated solution of S. cervisiae is added in the carrier that is pre-mixed, the weight ratio of concentrated solution and carrier is 0.6～0.9: 1, fluidised bed drying then, and 45～60 ℃ of drying temperatures obtain S. cervisiae dry powder; Described carrier comprises: lightweight CaCO
330～50wt%, inferior powder 20～30wt%, Zein powder 18～20wt%.
8, microbe additive as claimed in claim 1, its universal liquor is that fermentation of bacillus liquid, lactobacillus fermentation liquid, yeast fermentation broth are cultivated used substratum and the culture condition of rejuvenation according to flat board separately, viable bacteria content is carried out conventional plate count to be detected, according to count results, utilize aqua sterilisa that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml or 10
10CFU/ml, formulated according to following volume ratio then:
Fermentation of bacillus subtilis liquid 10～30v%; Bacillus cereus fermented liquid 10～30v%;
Lactobacillus fermentation liquid 30～50v%; Yeast saccharomyces cerevisiae fermented liquid 10～30v%.
9, microbe additive as claimed in claim 1, its young animal tailored version liquor is that fermentation of bacillus liquid, lactobacillus fermentation liquid, yeast fermentation broth are cultivated used substratum and the culture condition of rejuvenation according to flat board separately, viable bacteria content is carried out conventional plate count to be detected, according to count results, utilize aqua sterilisa that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml or 10
10CFU/ml, formulated according to following volume ratio then:
Fermentation of bacillus subtilis liquid 10～20v%; Bacillus cereus fermented liquid 10～20v%;
Lactobacillus fermentation liquid 40～60v%; Yeast saccharomyces cerevisiae fermented liquid 15～25v%.
10, microbe additive as claimed in claim 1, wherein large animal tailored version liquor is that fermentation of bacillus liquid, lactobacillus fermentation liquid, yeast fermentation broth are cultivated used substratum and the culture condition of rejuvenation according to flat board separately, viable bacteria content is carried out conventional plate count to be detected, according to count results, utilize aqua sterilisa that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml or 10
10CFU/ml, formulated according to following volume ratio then:
Fermentation of bacillus subtilis liquid 20～40v%; Bacillus cereus fermented liquid 15～30v%;
Lactobacillus fermentation liquid 20～40v%; Yeast saccharomyces cerevisiae fermented liquid 10～25v%.
11, microbe additive as claimed in claim 1, its ruminating animal tailored version liquor is that fermentation of bacillus liquid, lactobacillus fermentation liquid, yeast fermentation broth are cultivated used substratum and the culture condition of rejuvenation according to flat board separately, viable bacteria content is carried out conventional plate count to be detected, according to count results, utilize aqua sterilisa that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml or 10
10CFU/ml, formulated according to following volume ratio then:
Fermentation of bacillus subtilis liquid 15～30v%; Bacillus cereus fermented liquid 10～30v%;
Lactobacillus fermentation liquid 30～40v%; Yeast saccharomyces cerevisiae fermented liquid 15～35v%.
12, microbe additive as claimed in claim 1, its granulation feed tailored version liquor is that fermentation of bacillus liquid, lactobacillus fermentation liquid, yeast fermentation broth are cultivated used substratum and the culture condition of rejuvenation according to flat board separately, viable bacteria content is carried out conventional plate count to be detected, according to count results, utilize aqua sterilisa that viable bacteria content is diluted to 10 respectively
8CFU/ml, 10
9CFU/ml or 10
10CFU/ml, formulated according to following volume ratio then:
Fermentation of bacillus subtilis liquid 30～40v%; Bacillus cereus fermented liquid 20～30v%;
Lactobacillus fermentation liquid 20～30v%; Yeast saccharomyces cerevisiae fermented liquid 10～20v%.
13, microbe additive as claimed in claim 1, its universal pulvis is that genus bacillus dry powder, lactobacillus dry powder are cultivated used substratum and the culture condition of rejuvenation according to flat board separately, viable bacteria content to dry powder carries out conventional plate count detection, according to count results, utilize carrier that viable bacteria content is diluted to 10 respectively
8CFU/g, 10
9CFU/g or 10
10CFU/g, described carrier proportioning is: lightweight CaCO
325～50wt%, inferior powder 20～30wt%, Zein powder 15～20wt% form than preparation according to following weight then:
Subtilis dry powder 10～30wt%; Bacillus cereus dry powder 10～30wt%;
Lactobacillus dry powder 30～50wt%; S. cervisiae dry powder 10～30wt%.
14, microbe additive as claimed in claim 1, its young animal tailored version pulvis is that genus bacillus dry powder, lactobacillus dry powder are cultivated used substratum and the culture condition of rejuvenation according to flat board separately, viable bacteria content to dry powder carries out conventional plate count detection, according to count results, utilize carrier that viable bacteria content is diluted to 10 respectively
8CFU/g, 10
9CFU/g or 10
10CFU/g, described carrier proportioning is: lightweight CaCO
325～50wt%, inferior powder 20～30wt%, Zein powder 15～20wt% form than preparation according to following weight then:
Subtilis dry powder 10～20wt%; Bacillus cereus dry powder 10～20wt%;
Lactobacillus dry powder 40～60wt%; S. cervisiae dry powder 15～25wt%.
15, microbe additive as claimed in claim 1, wherein above the average age for marriage animal specific type pulvis is that genus bacillus dry powder, lactobacillus dry powder are cultivated used substratum and the culture condition of rejuvenation according to flat board separately, viable bacteria content to dry powder carries out conventional plate count detection, according to count results, utilize carrier that viable bacteria content is diluted to 10 respectively
8CFU/g, 10
9CFU/g or 10
10CFU/g, described carrier proportioning is: lightweight CaCO
325～50wt%, inferior powder 20～30wt%, Zein powder 15～20wt% form than preparation according to following weight then:
Subtilis dry powder 20～40wt%; Bacillus cereus dry powder 15～30wt%;
Lactobacillus dry powder 20～40wt%; S. cervisiae dry powder 10～25wt%.
16, microbe additive as claimed in claim 1, its ruminating animal tailored version pulvis is that genus bacillus dry powder, lactobacillus dry powder are cultivated used substratum and the culture condition of rejuvenation according to flat board separately, viable bacteria content to dry powder carries out conventional plate count detection, according to count results, utilize carrier that viable bacteria content is diluted to 10 respectively
8CFU/g, 10
9CFU/g or 10
10CFU/g, described carrier proportioning is: lightweight CaCO
325～50wt%, inferior powder 20～30wt%, Zein powder 15～20wt% form than preparation according to following weight then:
Subtilis dry powder 15～30wt%; Bacillus cereus dry powder 10～30wt%;
Lactobacillus dry powder 30～40wt%; S. cervisiae dry powder 15～35wt%.
17, microbe additive as claimed in claim 1, its granulation feed tailored version pulvis is that genus bacillus dry powder, lactobacillus dry powder are cultivated used substratum and the culture condition of rejuvenation according to flat board separately, viable bacteria content to dry powder carries out conventional plate count detection, according to count results, utilize carrier that viable bacteria content is diluted to 10 respectively
8CFU/g, 10
9CFU/g or 10
10CFU/g, described carrier proportioning is: lightweight CaCO
325～50wt%, inferior powder 20～30wt%, Zein powder 15～20wt% form than preparation according to following weight then:
Subtilis dry powder 30～40wt%; Bacillus cereus dry powder 20～30wt%;
Lactobacillus dry powder 20～30wt%; S. cervisiae dry powder 10～20wt%.
18, the described microorganism feed addictive of a kind of claim 1 is as the purposes of microbiotic substitute or product enzyme probiotics.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100311967A CN1281147C (en) | 2004-04-14 | 2004-04-14 | Microbial fodder additive and its preparing method and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100311967A CN1281147C (en) | 2004-04-14 | 2004-04-14 | Microbial fodder additive and its preparing method and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1682599A true CN1682599A (en) | 2005-10-19 |
| CN1281147C CN1281147C (en) | 2006-10-25 |
Family
ID=35262253
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100311967A Expired - Lifetime CN1281147C (en) | 2004-04-14 | 2004-04-14 | Microbial fodder additive and its preparing method and use |
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| Country | Link |
|---|---|
| CN (1) | CN1281147C (en) |
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