CN1281147C - Microbial fodder additive and its preparing method and use - Google Patents
Microbial fodder additive and its preparing method and use Download PDFInfo
- Publication number
- CN1281147C CN1281147C CNB2004100311967A CN200410031196A CN1281147C CN 1281147 C CN1281147 C CN 1281147C CN B2004100311967 A CNB2004100311967 A CN B2004100311967A CN 200410031196 A CN200410031196 A CN 200410031196A CN 1281147 C CN1281147 C CN 1281147C
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- bacillus
- fermentation
- liquid
- dry powder
- cfu
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to a microbial feed additive, particularly to a microbial feed additive which is composed of various beneficial microbes and metabolites, such as proteinase, lipase, amylase, cellulase, etc., a preparation method thereof and the application thereof. The microbial feed additive is the mixture of fermentation liquid or dry powder which prepared by that bacillus subtilis, bacillus cereus, lactobacillus and saccharomycete for brewing wine are respectively cultured by liquid submerged and high-density fermentation, and the feed additive is mixed and prepared into universal liquid or powder, or special liquid or powder for young animals, middle and old-age animals, or ruminants (cattle, sheep, etc.), or special liquid or powder for granular feed according to practical purposes. The microbial feed additive has the advantages of high active bacterium content, long product retention time and little contamination by foreign bacteria. The microbial feed additive can improve feed utilization rates, promote animal growth, increase the yield of meat, eggs, milk, etc., improve the balance of intestinal flora or enzymes, improve immunity, disease resistance capability, replace antibiotic, etc., and improve animal product quality.
Description
Technical field
The present invention relates to a kind of additive for microbe feedstuff, specifically relate to a kind of additive for microbe feedstuff that is formed by multiple beneficial microorganism and its metabolite (such as protease, lipase, amylase, cellulase etc.) and its production and use.
Background technology
The use of some growth promotion health-care agents has promoted the fast development of animal husbandry in feed industry and the aquaculture, but has brought many potential threats for again simultaneously the development of human health and feed industry. The growth promotion 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, sedative, cause series of problems and consequence: (1) causes the animal products drug residue serious, directly be detrimental to health, even generation " three cause ", and make the animal products of China and aquatic products when entering the international market, face thick-and-thin, suffer huge economic loss every year. (2) cause the animal products quality to descend, lack the market competitiveness and trust. Widely used p-aminophenyl arsine (arsenic) acid waits arsenic preparation in feed at present, is classified as carcinogen by international cancer research institution (IARC, 1980). In addition, at the medium-term and long-term sedative etc. that uses of pig industry, cause medicament residue. China Ministry of Public Health report (1998), annual newly-increased 2,000,000 cancer patients of China, wherein 1,000,000 people die. This makes the consumer produce " sitophobia " to animal products. (3) cause bacterial drug resistance enhancing, antibody-resistant bacterium to increase, as a result medication 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 to the action of a drug germs that livestock produces, can pass to the people by food, and produces the disease that is difficult to healing, and harm humans is healthy. Simultaneously, the antibiotic that aureomycin, terramycin, chloramphenicol, olaquindox, sulfonamide, Madumycin etc. are easy residual and toxicity is larger and a large amount of uses of synthesising bacteria anti-reflecting medicine, cause medicament residue to increase, the antibody-resistant bacterium diffusion, the animal flora imbalance, the immunity of animal is suppressed, the secondary superinfection. (4) cause environmental destruction. Environmental protection becomes the problem that the people of other countries more and more are concerned about just day by day. The pollution that intensive Production of Livestock and Poultry causes has become the key factor of restriction Production of Livestock and Poultry. According to EEC report, European nitrous oxide (N2O) and have respectively 33% and 34% in the methane emission from its feeding field. Nitrous oxide can form nitrogen dioxide under photochemical effect, again can with ozonization, can cause cutaneum carcinoma. In the intensive culture, livestock and poultry are discharged a large amount of ammoniacal nitrogens and phosphorus etc., and ammonia is the very strong material of a kind of contaminative, affect the growth of plant by changing the water and soil Acidity of Aikalinity. In addition, 70% phosphorus that will take in the body drains nonruminant owing to can't utilize phytate phosphorus, and the 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 growth promotion health-care agent have more than 1000 veterinary drug enterprises production and sales 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 subject to consumer's generally opposition, in recent years, the consumer pays attention to safety, health and the health of food especially, formed the common recognition of " mankind of the food of healthy animal → safety → health ", 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 antibiotic separately, all forbade antibiotic from 2006 such as European Union, the World Health Organization (WHO) has proposed " reducing the global principle that antibiotic uses in edible animal ", China starts " feed safety engineering ", and has forbidden a collection of toxicity and residual larger medicine. Face the consumer of countries in the world to the strong request of food " safety, healthy, nutrition " and back to nature, every country is just at active development green feed additive or green growth promotion health-care agent. Additive for microbe feedstuff or claim little ecological active bacteria formulation, probiotic, 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 human body immune function, reduce mortality of animals, improve food conversion ratio and the advantage such as animal feeding benefit that the extensive approval and the parent that are subject to countries in the world scientist and animal feeding person look at. Using microbe feed addictive in feed or animal feeding; alternative antibiotic or significantly subdue the consumption of antibiotic etc; improve the quality of animal products and the international competitiveness of local flavor and enhancing animal products; be conducive to continuing and sound development of animal husbandry; be conducive to the health of animal and human's class, be conducive to environmental protection. Therefore, development and production micro organism green feed addictive has important reality, ecology and economic implications.
Additive for microbe feedstuff, be referred to as in the world probiotic (Probiotics), its general definition refer to contain viable bacteria and (or) dead bacterium, the bacterial preparation that comprises its component and product, per os or drop into via other mucous membrane approach, be intended to improve the balance of mucomembranous surface microorganism or enzyme, perhaps stimulate specificity or nonspecific immunity mechanism (1994, more than 40 up-to-date definition that country participates in the German international conference).
Bacillus acidi lactici, saccharomycete and bacillus be have separately Biological characteristics and the probio class of function. Wherein, Bacillus acidi lactici 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 alimentary canal, " be throughout one's life the companion; be throughout one's life benefit " with animal, bringing into play antagonism pathogenic microorganism (such as Escherichia coli and salmonella etc.), start special and nospecific immunity system (strengthening immunity and premunition), reduce cholesterol, in and enterotoxin, synthesize nutriment, produce digestive enzymes and regulate the function such as raising poultry nutritive metabolism. Saccharomycete is the oxygen consumption microorganism, and it can not only help animal digestion, can also promote the endogenous vigor that beneficial microorganism is arranged of alimentary canal, replenishes B family vitamin, and its cell wall polysaccharides also has stimulating animal immunity, improves the effect of premunition. Benefit is given birth to the type bacillus; belong to the oxygen consumption type microorganism; they can be grown rapidly, grow after entering alimentary canal; produce very strong protease, lipase, amylase, cellulase isoreactivity material; important regulating and controlling effect is being brought into play in digestion, absorption and utilization to nutriments such as protein, fat, carbohydrate; can also produce Cobastab and vitamin K; by reducing oxidation-reduction potential in the alimentary canal; and produce a small amount of antibacterial material; come harmful microorganism is played inhibition and killing action; simultaneously; its spore has very strong tolerance to poor environments such as heat, pressure, acid, alkali, enzyme and some drugs, X ray; good stability in feed processing, storage and alimentary canal; need not the special protection processing; just can survive for a long time, therefore, can be widely used in domestic animal, fowl and the aquatic livestocks such as pig, ox, chicken.
At present, some Feed Enterprise are all at the additive for microbe feedstuff of actively seeking and study new environmental protection. But this type of additive for microbe feedstuff on the market generally mostly is a kind of single bacteria preparation that makes that uses in Bacillus acidi lactici, saccharomycete and the bacillus, function singleness, viable bacteria content is low in the product, production technology and equipment are more backward, the living bacterial cells time-to-live is short, inferior quality, and living contaminants is serious, and result of use is undesirable, unstable.
Summary of the invention
The object of the invention is to utilize the reasonable compound compatibility of the useful functional microorganism of three classes, 4 strains, employing liquid deep layer fermenting technology and a series of after-processing technology technology that comprise Bacillus acidi lactici, saccharomycete and bacillus, overcome in existing additive for microbe feedstuff function singleness, the product that viable bacteria content is low, the living bacterial cells time-to-live short, the product living contaminants is serious, result of use is undesirable, the unsettled defective of effect, thereby provide that a kind of viable bacteria content is high, the product holding time is long, living contaminants is few, can improve the additive for microbe feedstuff of feed nutrient and efficiency of feed utilization.
Another object of the present invention is to provide the preparation method of described additive for microbe feedstuff.
A further object of the present invention is to provide the purposes of described additive for microbe feedstuff.
The objective of the invention is to realize by the following technical solutions:
The invention provides a kind of additive for microbe feedstuff, its liquor type is cultivated for bacillus subtilis (Bacillus subtilis), Bacillus cercus (Bacillus cereus), Bacillus acidi lactici (Lactobacillus sp.) and S. cervisiae (Saccharomyces cerevisiae) being carried out respectively liquid deep layer fermenting, and the zymotic fluid of each single bacterium mixed and get, the viable count of bacillus subtilis+Bacillus cercus+Bacillus acidi lactici+S. cervisiae has three specifications in the zymotic fluid that mixes, namely 〉=100,000,000/ml, 〉=10 hundred million/ml and 〉=100 hundred million/ml.
The liquid submerged femrentation culturing method of the zymotic fluid of described bacillus subtilis (Bacillus subtilis) and Bacillus cercus (Bacillus cereus) comprises following step:
1) dull and stereotypedly cultivates rejuvenation: bacillus subtilis and cured shape bacillus bacterial classification are adopted respectively method streak inoculation on the bacillus plating medium of streak inoculation, cultivated 12～24 hours in 25～35 ℃, make respectively bacillus subtilis and the rejuvenation of cured shape bacillus, and form single bacterium colony; Described bacillus plating medium is the mixture by following w/v (W/V) preparation: peptone 0.5～1%, glucose 0.5～2%, beef extract 0.5～1.5%, potassium dihydrogen phosphate 0.3～0.6%, agar 3～4%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, for subsequent use;
2) choose respectively single bacterium colony the bacillus subtilis of making shake-flask seed: from step 1) cultivating and the cured shape bacillus bacterium colony, be inoculated in the bacillus liquid culture medium, in 28～37 ℃ of cultivations 20～30 hours, obtain bacillus subtilis and cured shape bacillus shake-flask seed liquid; Described bacillus liquid culture medium is the mixed liquor by following w/v (W/V) preparation: peptone 0.5～1%, glucose 0.5～2%, beef extract 0.5～1.5%, potassium dihydrogen phosphate 0.3～0.6%, 3.08% manganese sulfate solution 0.5～1.5%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, for subsequent use;
3) the bacillus subtilis shake-flask seed liquid that production primary seed solution: with step 2) obtains and cured shape bacillus shake-flask seed liquid are with the inoculum concentration of 2～5v%, be inoculated in respectively in the first order seed liquid culture medium of producing used first class seed pot, cultivate 18～24h in 32～37 ℃, mixing speed 150～200rpm, throughput is that 1～2vols/vol/min (is abbreviated as: vvm), obtain respectively the primary seed solution of bacillus subtilis and Bacillus cercus; Described first order seed liquid culture medium prescription is the mixed liquor by following w/v (W/V) preparation: 120 order bean cake powders 0.5～2.0%, 120 order fish meal 0.2～1.5%, 120 order corn flours 0.5～1.0%, glucose 1.0～4.5%, NaCl 0.1～0.3%, NaOH 0.05～0.10%, Na2HPO
40.1～0.5%, synergist 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 synergist is open in first to file 200410008993.3, for by MgSO40.1～0.5g/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) bacillus subtilis and the cured shape bacillus first order seed that second order fermentation (producing fermentation): with step 3) obtain, be inoculated into 5～10% volume ratio respectively and be equipped with in the fermentation tank of producing fermentation culture, prescription and the controlled condition of fermentation culture are same as step 3) first order seed liquid culture medium, ferment after 16～24 hours, by microscopic examination, when being arranged, a large amount of sporulation stops fermentation.
When industrial production, when the output of needs more extensive (fermentation tank more than 20 tons), also comprise step 5) carry out three grade fermemtation:
5) bacillus subtilis that three grade fermemtation: with step 4) obtains and cured shape bacillus secondary seed; ratio with 5～10% volumes is inoculated in the fermentation tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and controlled condition; ferment after 16～24 hours; by microscopic examination; when a large amount of sporulation is arranged, shut down.
The cultural method of the zymotic fluid of described Bacillus acidi lactici (Lactobacillus sp.) comprises following step:
1) dull and stereotyped cultivation rejuvenation: with method streak inoculation on the Bacillus acidi lactici plating medium of Bacillus acidi lactici inclined-plane seed employing streak inoculation, in 33～38 ℃ of cultivations 24～48 hours, make the Bacillus acidi lactici rejuvenation, and form single bacterium colony; Described Bacillus acidi lactici plating medium is the mixture by following w/v (W/V) preparation: soy peptone 0.5～1%, glucose 1～2%, yeast soak powder 0.3～0.7%, potassium dihydrogen phosphate 0.3～0.5%, beef extract 0.5～1.5%, Triammonium citrate 1.5～2.3%, Tween-800.5～1.4%, sodium acetate 0.2～0.6%, agar powder 2～2.5%, pH=5.5～5.8, sterilized 15 minutes for 121 ℃, for subsequent use;
2) choose single bacterium colony in the Bacillus acidi lactici bacterium colony of making shake-flask seed: with step 1) cultivating, be inoculated in the Bacillus acidi lactici fluid nutrient medium, the employing conventional method in 35～40 ℃ of cultivations 24～48 hours, obtains the Bacillus acidi lactici shake-flask seed on the triangular flask fluid nutrient medium; Described culture medium is the mixed liquor by following w/v (W/V) preparation: soy peptone 0.5～1%, glucose 1～2%, yeast soak powder 0.3～0.7%, potassium dihydrogen phosphate 0.3～0.5%, beef extract 0.5～1.5%, Triammonium citrate 1.5～2.3%, Tween-800.5～1.4%, sodium acetate 0.2～0.6%, pH=5.5～5.8, sterilized 15 minutes for 121 ℃, for subsequent use;
3) the Bacillus acidi lactici shake-flask seed that production primary seed solution: with step 2) obtains is with the inoculum concentration of 6～12v%, be inoculated in the first order seed liquid culture medium of producing used first class seed pot, cultivate 17～60h in 35～38 ℃, mixing speed 150～200rpm, interval ventilation in 1 hour 5～10 minutes, throughput is 1～2.5vols/vol/min (vvm), until the Bacillus acidi lactici number reaches 1,000,000,000, obtains the primary seed solution of Bacillus acidi lactici; Described first order seed liquid culture medium prescription is the mixed liquor by following w/v (W/V) preparation: 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 corn flours 0.5～1.0%, glucose 1～2.5%, NaCl0.1～0.3%, Na2HPO
40.15～0.3%, synergist 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 synergist is open in first to file 200410008992.9, for by CaCl22～4g/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) the Bacillus acidi lactici first order seed that second order fermentation (producing fermentation): with step 3) obtains, be inoculated in the fermentation tank that the production fermentation culture is housed with 5～10% volume ratios, prescription and the controlled condition of fermentation culture are same as step 3) first order seed liquid culture medium, 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 Bacillus acidi lactici amount reaches greater than 109The CFU/ml zymotic fluid) stops fermentation.
When industrial production, when the output of needs more extensive (fermentation tank more than 20 tons), also comprise step 5) carry out three grade fermemtation:
5) the Bacillus acidi lactici secondary seed that three grade fermemtation: with step 4) obtains; ratio with 5～10% volumes is inoculated in the fermentation tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and controlled condition; ferment after 17～60 hours; detect until the Bacillus acidi lactici number reaches 1,000,000,000/ml, shut down.
The cultural method of the zymotic fluid of described saccharomyces cerevisiae (Saccharomyces cerevisiae) comprises following step:
1) dull and stereotyped cultivation rejuvenation: with method streak inoculation on the saccharomyces cerevisiae plating medium of saccharomyces cerevisiae inclined-plane seed employing streak inoculation, in 24～30 ℃ of cultivations 20～24 hours, make the saccharomyces cerevisiae rejuvenation, and form single bacterium colony; Described saccharomyces cerevisiae plating medium is the mixture by following w/v (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 ℃, for subsequent use;
2) choose single bacterium colony the saccharomyces cerevisiae culture plate of making shake-flask seed: from step 1) cultivating, be inoculated in the triangular flask liquid medium, in 28～32 ℃ of cultivations 24～37 hours, obtain the saccharomyces cerevisiae shake-flask seed liquid; Described nutrient solution is the mixed liquor by following w/v (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%, pH=5.8～6.0, sterilized 15 minutes for 121 ℃, for subsequent use;
3) the saccharomyces cerevisiae shake-flask seed that production primary seed solution: with step 2) obtains is by the inoculum concentration of 2～12v%, be inoculated in the first order seed nutrient solution of first class seed pot, cultivated 24～37 hours in 28～32 ℃, mixing speed 160～220rpm, throughput is 1.0～3.0vols/vol/min (vvm), obtains the primary seed solution of saccharomyces cerevisiae; Described saccharomyces cerevisiae first order seed nutrient solution is the mixed liquor by following w/v (W/V) preparation: 120 order bean cake powders 0.5～1.5%, 120 order fish meal 0.2～0.8%, 120 order corn flours 0.5～1.0%, glucose 0.3～1.0%, NaCl0.1～0.3%, Na2HPO
40.2～0.5%, synergist 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 synergist is open in first to file 200410008994.8, for by MgSO40.05～0.3g/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) the saccharomyces cerevisiae first order seed that second order fermentation (producing fermentation): with step 3) obtains, be inoculated in the fermentation tank that the production fermentation culture is housed with 4～10% volume ratios, prescription and the controlled condition of fermentation culture are same as step 3) the first order seed nutrient solution, ferment after 24～60 hours, detection S. cervisiae number stops fermentation when reaching 1,000,000,000/ml.
When industrial production, when the output of needs more extensive (fermentation tank more than 20 tons), also comprise step 5) carry out three grade fermemtation:
5) the saccharomyces cerevisiae secondary seed that three grade fermemtation: with step 4) obtains; ratio with 5～10% volumes is inoculated in the fermentation tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and controlled condition; ferment after 24～60 hours; detect until the S. cervisiae number reaches 1,000,000,000/ml, shut 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 culture medium and the condition of culture 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 respectively 108CFU/ml、10
9CFU/ml or 1010CFU/ml, then formulated according to following volume ratio according to practical use:
The universal proportioning of liquor type microbe additive is:
Fermentation of bacillus subtilis liquid 10～30v%; Bacillus cereus fermentation liquid 10～30v%;
Lactobacillus fermentation liquid 30～50v%; Saccharomyces cerevisiae fermented liquid 10～30v%.
The proportioning of liquor type microbe additive children animal specific type in age is:
Fermentation of bacillus subtilis liquid 10～20v%; Bacillus cereus fermentation liquid 10～20v%;
Lactobacillus fermentation liquid 40～60v%; 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 fermentation liquid 15～30v%;
Lactobacillus fermentation liquid 20～40v%; Saccharomyces cerevisiae fermented liquid 10～25v%.
The proportioning of liquor type microbe additive ruminant (cattle and sheep etc.) tailored version is:
Fermentation of bacillus subtilis liquid 15～30v%; Bacillus cereus fermentation liquid 10～30v%;
Lactobacillus fermentation liquid 30～40v%; 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 fermentation liquid 20～30v%;
Lactobacillus fermentation liquid 20～30v%; Saccharomyces cerevisiae fermented liquid 10～20v%.
Additive for microbe feedstuff provided by the invention, its powder-type is for to make dry powder with the zymotic fluid of above-mentioned bacillus subtilis, Bacillus cercus, Bacillus acidi lactici and S. cervisiae through different after-processing technologies, mixture in following ratio preparation, the viable count of bacillus subtilis+Bacillus cercus in the dry powder+Bacillus acidi lactici+S. cervisiae has three specifications, namely 〉=100,000,000/ml, 〉=10 hundred million/ml and 〉=100 hundred million/ml.
The preparation method of described bacillus subtilis dry powder and Bacillus cercus dry powder comprises following step:
With the zymotic fluid of bacillus subtilis and the zymotic fluid of Bacillus cercus, be that 0.5～0.8: 1 ratio mixes according to the weight ratio of zymotic fluid 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 respectively bacillus subtilis dry powder and Bacillus cercus dry powder; Described carrier (adsorbent) proportioning is: lightweight CaCO325～50wt%, inferior powder 20～30wt%, corn protein powder 15～20wt%;
Or the zymotic fluid of the zymotic fluid of bacillus subtilis and Bacillus cercus by volume added the dextrin of fermentating liquid volume 0.3～0.8% and 3～10% precipitated calcium carbonate with weight ratio respectively, after mixing, press 100～130 ℃ of air intakes at spray dryer, 60～90 ℃ of air-out, 2kg is pressed in spray, flow 1000～1500ml/min carries out spray-drying, obtains bacillus subtilis spraying dry powder and Bacillus cercus spraying dry powder.
The preparation method of described dry powder of lactic acid bacteria comprises following step:
1) zymotic fluid with above-mentioned lactic acid bacteria is concentrated to 40～60% of original volume through hot low temperature (＜45 ℃) negative-pressure vacuum;
2) adding accounts for the protective agent of the weight/percent by volume 15～20% of concentrate, in-25～-45 ℃ of freeze dryings, obtains the lactic acid bacteria freeze drying powder after mixing; Described protective agent be skimmed milk power, white granulated sugar (sucrose), maltodextrin, gelatin in 1: 2: 1: 1 ratio mixes;
Or the concentrate of lactic acid bacteria added in the carrier that is pre-mixed, the weight ratio of concentrate and carrier is 0.4～0.9: 1, then through 45～60 ℃ of dryings of fluid bed, is crushed to more than 80 orders, obtains dry powder of lactic acid bacteria; Described carrier comprises: lightweight CaCO330～40wt%, inferior powder 15～30wt%, corn protein powder 18～20wt%.
The preparation method of described S. cervisiae dry powder comprises following step:
1) zymotic fluid with above-mentioned S. cervisiae is concentrated to 40～60% of original volume through hot low temperature (＜45 ℃) negative-pressure vacuum, obtains the concentrate of S. cervisiae;
2) concentrate of S. cervisiae is added in the carrier that is pre-mixed, the weight ratio of concentrate and carrier is 0.6～0.9: 1, fluidized bed drying then, and 45～60 ℃ of baking temperatures obtain S. cervisiae dry powder; Described carrier comprises: lightweight CaCO330～50wt%, inferior powder 20～30wt%, corn protein powder 18～20wt%.
Microbe additive pulvis provided by the invention is that above-mentioned bacillus dry powder, lactobacillus dry powder are cultivated used culture medium and the condition of culture 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 respectively 108CFU/g、10
9CFU/g or 1010CFU/g, described carrier proportioning is: lightweight CaCO3Then 25～50wt%, inferior powder 20～30wt%, corn protein powder 15～20wt% form than preparation according to following weight according to practical use:
The proportioning that the powder-type microbe additive is universal:
Bacillus subtilis dry powder 10～30wt%; Bacillus cercus dry powder 10～30wt%;
Lactobacillus dry powder 30～50wt%; S. cervisiae dry powder 10～30wt%.
The proportioning of powder-type microbe additive children animal specific type in age is:
Bacillus subtilis dry powder 10～20wt%; Bacillus cercus 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:
Bacillus subtilis dry powder 20～40wt%; Bacillus cercus dry powder 15～30wt%;
Lactobacillus dry powder 20～40wt%; S. cervisiae dry powder 10～25wt%.
The proportioning of powder-type microbe additive ruminant (cattle and sheep etc.) tailored version is:
Bacillus subtilis dry powder 15～30wt%; Bacillus cercus 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:
Bacillus subtilis dry powder 30～40wt%; Bacillus cercus dry powder 20～30wt%;
Lactobacillus dry powder 20～30wt%; S. cervisiae dry powder 10～20wt%.
The quality standard of mentioned microorganism feed addictive provided by the invention is as follows:
Liquor: the viable bacteria summation of 4 kinds of microorganisms 〉=108/ milliliter (100,000,000), 109/ milliliter (1,000,000,000) and 1010/ milliliter (10,000,000,000) 3 kinds of models or specification, equal brown color liquid, denseer sour fragrance; Acidic liquid, pH≤4.5.
Pulvis: the viable bacteria summation of 4 kinds of microorganisms 〉=100,000,000/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 additive for microbe feedstuff. This additive is as Substitutes For Antibiotic or produce enzyme probiotic (enzyme-added probiotic), eliminate medicament residue, improve the 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 immunity, premunition and feed efficiency, reduction animal death rate, also can be used for making ensiling, yellow storage feed and biological feedstuff, 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.
Additive for microbe feedstuff provided by the invention (Yishengkang) mainly is comprised of protease, lipase, amylase, cellulolytic enzyme and the Substance of safe and efficient useful Bacillus, lactic acid bacteria, saccharomycete etc. and microorganisms, selected bacterial classification belongs to the microbe species of 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 ecological culturing, be non-harmful green feed additive. The characteristics that additive for microbe feedstuff provided by the invention utilizes saccharomycete and bacillus can consume dissolved oxygen in the intestines and stomach food, for having beneficial microorganism such as lactic acid bacteria class, Bifidobacterium etc., the endogenous anaerobism provides anaerobic environment, anaerobic environment can suppress to like the growth and breeding of the harmful microorganism of oxygen such as Escherichia coli and salmonella etc. simultaneously, thereby safeguard the balance of microbial population of animal intestinal tract and the health of animal, brought into play simultaneously the function of three class beneficial bacteriums and intestines endogenous beneficial bacterium, finally played growth promotion, 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 additive for microbe feedstuff 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, viable bacteria content is high, and production efficiency is high, and the viable bacteria in 3 kinds of specification products of liquor can reach 10 respectively8/ milliliter, 109/ milliliter or 1010/ milliliter, the viable bacteria in 3 kinds of specification products of pulvis also can reach 108/g、10
9/ g or 1010/ 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 not thoroughly and during the fermentation miscellaneous bacteria breeding of sterilization, cause harmful bacteria and object bacteria (beneficial bacterium) to be mixed together growth, the problem such as product is impure; The 2nd, the present invention is in aftertreatment technology, to carrier or adsorbent first through fluidizing drying bed at 105 ℃, 1～2 minute dry sterilization is processed, kill some harmful bacterias in the carrier raw material, so that the object bacteria composition purity of product is high, clear efficacy, stable can not affect because of the existence of miscellaneous bacteria the effect of product;
4) additive for microbe feedstuff of the present invention is 4 kinds of beneficial bacteriums, the science combination, and the ratio collocation is proper rationally, and multiple functional, clear and definite, effect is strong;
5) add during the fermentation synergist, made total amount of probiotics improve several times to tens times than existing product, and prolonged the storage life of product, improved the stable effect of product;
6) this method low cost product of producing is suitable for being widely used in production of fodder and herding, aquaculture, has significant economic benefit, social benefit and ecological benefits.
The specific embodiment
The zymotic fluid (A) of embodiment 1, cultivation bacillus subtilis
1) dull and stereotypedly cultivates rejuvenation: the preservation of bacteria strain of bacillus subtilis is adopted method streak inoculation on the bacillus plating medium of streak inoculation, cultivated 18 hours in 30 ℃, make the bacillus 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, down together), glucose 0.5%, beef extract 0.5%, potassium dihydrogen phosphate 0.3%, agar 3%, pH=7.5～7.6,121 ℃ sterilization 30 minutes, for subsequent use;
2) select typical single bacterium colony the bacillus subtilis bacterium colony of making shaking flask (triangular flask) seed: from step 1) cultivating, be inoculated in the bacillus liquid culture medium, in 32 ℃ of cultivations 30 hours, obtain the bacillus subtilis shake-flask seed liquid; Described bacillus liquid culture medium is the mixed liquor in following ratio preparation: peptone 0.5%, glucose 2%, beef extract 0.5%, potassium dihydrogen phosphate 0.6%, 3.08% manganese sulfate solution 1.0%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, for subsequent use;
3) production primary seed solution: first according to formulated first order seed liquid culture medium described later, pH=7.5～7.6, through 121 ℃, sterilized 45 minutes, be cooled to 37 ℃, then with step 2) the bacillus subtilis shake-flask seed liquid that obtains is with the inoculum concentration of 2v%, be inoculated in respectively and produce in the used first class seed pot, cultivate 24h, mixing speed 200rpm in 37 ℃, throughput is 2vvm, obtains the primary seed solution of bacillus subtilis; Described first order seed liquid culture medium prescription is the mixed liquor (W/V) in following ratio preparation, 120 order bean cake powders 1.0%, 120 order fish meal 1.0%, 120 order corn flours 0.5%, glucose 2.0%, NaCl 0.2%, NaOH 0.08%, Na2HPO
40.3%, synergist 0.1%, defoamer 0.08%; Described synergist is open in first to file 200410008993.3, for by MgSO40.1～
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) the bacillus subtilis first order seed that second order fermentation (producing fermentation): with step 3) obtains; ratio with 10% volume is inoculated in the fermentation tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and controlled condition; ferment after 20 hours; by microscopic examination; when a large amount of sporulation is arranged, shut down, obtain the zymotic fluid of bacillus subtilis. At last according to dull and stereotyped used culture medium and the condition of culture of rejuvenation of cultivating of bacillus 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 running water that viable bacteria content is diluted to respectively 108CFU/ml、10
9CFU/ml and 1010CFU/ml obtains the reserve liquid (A) of fermentation of bacillus subtilis liquid, treats post processing.
The zymotic fluid (A) of embodiment 2, cultivation bacillus subtilis
1) dull and stereotypedly cultivates rejuvenation: the preservation of bacteria strain of bacillus subtilis is adopted method streak inoculation on the bacillus plating medium of streak inoculation, cultivated 18 hours in 30 ℃, make the bacillus 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%, down together), glucose 0.5%, beef extract 0.5%, potassium dihydrogen phosphate 0.3%, agar 3%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, for subsequent use;
2) select typical single bacterium colony the bacillus subtilis bacterium colony of making shaking flask (triangular flask) seed: from step 1) cultivating, be inoculated in the bacillus liquid culture medium, in 32 ℃ of cultivations 30 hours, obtain the bacillus subtilis shake-flask seed liquid; Described bacillus liquid culture medium is the mixed liquor in following ratio preparation: peptone 0.5%, glucose 2%, beef extract 0.5%, potassium dihydrogen phosphate 0.6%, 3.08% manganese sulfate solution 1.0%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, for subsequent use;
3) production primary seed solution: first according to formulated first order seed liquid culture medium described later, pH=7.5～7.6, through 121 ℃, sterilized 45 minutes, be cooled to 37 ℃, then with step 2) the bacillus subtilis shake-flask seed liquid that obtains is with the inoculum concentration of 2v%, be inoculated in respectively and produce in the used first class seed pot, cultivate 24h, mixing speed 200rpm in 37 ℃, throughput is 2vvm, obtains the primary seed solution of bacillus subtilis; Described first order seed liquid culture medium prescription is the mixed liquor (W/V) in following ratio preparation, 120 order bean cake powders 1.0%, 120 order fish meal 1.0%, 120 order corn flours 0.5%, glucose 2.0%, NaCl0.2%, NaOH0.08%, Na2HPO
40.3%, synergist 0.1%, defoamer 0.08%; Described synergist is open in first to file 200410008993.3, for by MgSO40.1～
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) the bacillus subtilis first order seed that second order fermentation (producing fermentation): with step 3) obtains; ratio with 10% volume is inoculated in the fermentation tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and controlled condition; ferment after 16 hours; shut down, obtain the secondary seed of bacillus subtilis.
5) the bacillus subtilis secondary seed that three grade fermemtation: with step 4) obtains; ratio with 5% volume is inoculated in the three grade fermemtation tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and controlled condition; ferment after 20 hours; by microscopic examination; when a large amount of sporulation is arranged, shut down, obtain fermentation of bacillus subtilis liquid. At last according to dull and stereotyped used culture medium and the condition of culture of rejuvenation of cultivating of bacillus 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 running water that viable bacteria content is diluted to respectively 108CFU/ml、10
9CFU/ml and 1010CFU/ml obtains the reserve liquid (A) of fermentation of bacillus subtilis liquid, treats post processing.
The zymotic fluid (B) of embodiment 3, cultivation Bacillus cercus
Microbe species is Bacillus cercus (Bacillus cereus), culture medium, condition, preparation method are with embodiment 1, obtain bacillus cereus fermentation liquid, at last according to dull and stereotyped used culture medium and the condition of culture of rejuvenation of cultivating of Bacillus cereus, viable bacteria content to the Bacillus cereus zymotic fluid carries out conventional plate count detection, according to count results, utilize the sterilization running water that viable bacteria content is diluted to respectively 108CFU/ml、10
9CFU/ml and 1010CFU/ml obtains the reserve liquid (B) of bacillus cereus fermentation liquid, treats post processing.
The zymotic fluid (B) of embodiment 4, cultivation Bacillus cercus
1) microbe species is Bacillus cercus (Bacillus cereus), and culture medium, condition, preparation method obtain the Bacillus cercus secondary seed solution with embodiment 2.
2) the Bacillus cercus secondary seed that three grade fermemtation: with step 1) obtains; ratio with 8% volume is inoculated in the three grade fermemtation tank of fermentation culture; the prescription of fermentation culture and controlled condition are with step 2); ferment after 22 hours; by microscopic examination; when a large amount of sporulation is arranged; shut down; obtain bacillus cereus fermentation liquid; at last according to dull and stereotyped used culture medium and the condition of culture of rejuvenation of cultivating of Bacillus cereus; viable bacteria content to the Bacillus cereus zymotic fluid carries out conventional plate count detection, according to count results, utilizes the sterilization running water that viable bacteria content is diluted to respectively 108CFU/ml、10
9CFU/ml and 1010CFU/ml obtains the reserve liquid (B) of bacillus cereus fermentation liquid, treats post processing.
Embodiment 5, secondary are cultivated Bacillus acidi lactici (Lactobacillus sp.) zymotic fluid (C)
1) dull and stereotypedly cultivates rejuvenation: the Bacillus acidi lactici inclined-plane seed of preservation is adopted method streak inoculation on the Bacillus acidi lactici plating medium of streak inoculation, cultivated 24 hours in 33 ℃, make the Bacillus acidi lactici rejuvenation, and form single bacterium colony; Described Bacillus acidi lactici culture medium is the mixture in following ratio preparation: soy peptone 1%, glucose 2%, yeast soak powder 0.7wt%, potassium dihydrogen phosphate 0.3%, beef extract 0.5%, Triammonium citrate 2.3%, Tween-801.4%, sodium acetate 0.2%, agar powder 2%, pH=5.5～5.8,121 ℃, sterilized 15 minutes, for subsequent use;
2) select typical single bacterium colony in the Bacillus acidi lactici bacterium colony of making shake-flask seed: with step 1) cultivating, be inoculated in the Bacillus acidi lactici fluid nutrient medium, the employing conventional method in 35 ℃ of cultivations 30 hours, obtains the Bacillus acidi lactici shake-flask seed on the triangular flask fluid nutrient medium; Described culture medium is the mixed liquor in following ratio preparation: soy peptone 1%, glucose 1%, yeast soak powder 0.7wt%, potassium dihydrogen phosphate 0.5%, beef extract 0.5%, Triammonium citrate 1.5%, Tween-800.5%, sodium acetate 0.6%, pH=5.5～5.8,115 ℃, sterilized 15 minutes, for subsequent use;
3) production primary seed solution: first according to formulated first order seed liquid culture medium described later, through 121 ℃, sterilized 45 minutes, pH=5.5～5.8, be cooled to 37 ℃, then with step 2) the Bacillus acidi lactici shake-flask seed that obtains is with the inoculum concentration of 8v%, be inoculated in and produce in the used first class seed pot, cultivate 30h in 35 ℃, stir fast 150rpm, interval ventilation in 1 hour 5 minutes, throughput is 1.0vvm, until the Bacillus acidi lactici number reaches 1,000,000,000, obtain Bacillus acidi lactici and produce used primary seed solution; Described first order seed fermentation culture is the mixed liquor (W/V) in following ratio preparation: 120 order bean cake powders 0.5,120 order fish meal 0.5%, fish peptone 0.5%, lactose 0.2%, 120 order corn flour 0.5%, glucose 1%, NaCl0.1%, Na2HPO
40.15%, synergist 0.05%, defoamer 0.03%, pH=5.5～5.8; Described synergist is open in first to file 200410008992.9, for by CaCl22～4g/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 culture medium is same as step 2) in culture medium;
4) the Bacillus acidi lactici first order seed that second order fermentation (producing fermentation): with step 3) obtains, ratio with 10% volume is inoculated in the fermentation tank that the production fermentation culture is housed, the same step 3) of the prescription of fermentation culture and controlled condition, fermented 24 hours, obtain lactobacillus fermentation liquid, at last according to dull and stereotyped used culture medium and the condition of culture of rejuvenation of cultivating of Bacillus acidi lactici, viable bacteria content to lactobacillus fermentation liquid carries out conventional plate count detection, according to count results, utilize the sterilization running water that viable bacteria content is diluted to respectively 108CFU/ml、10
9CFU/ml and 1010CFU/ml obtains the reserve liquid (C) of lactobacillus fermentation liquid, treats post processing.
Embodiment 6, three grade fermemtation Bacillus acidi lactici liquid (C)
1) dull and stereotypedly cultivates rejuvenation: the Bacillus acidi lactici inclined-plane seed of preservation is adopted method streak inoculation on the Bacillus acidi lactici plating medium of streak inoculation, cultivated 24 hours in 33 ℃, make the Bacillus acidi lactici rejuvenation, and form single bacterium colony; Described Bacillus acidi lactici culture medium is the mixture in following ratio preparation: soy peptone 1%, glucose 2%, yeast soak powder 0.7wt%, potassium dihydrogen phosphate 0.3%, beef extract 0.5%, Triammonium citrate 2.3%, Tween-801.4%, sodium acetate 0.2%, agar powder 2%, pH=5.5～5.8,121 ℃, sterilized 15 minutes, for subsequent use;
2) select typical single bacterium colony in the Bacillus acidi lactici bacterium colony of making shake-flask seed: with step 1) cultivating, be inoculated in the Bacillus acidi lactici fluid nutrient medium, the employing conventional method in 35 ℃ of cultivations 30 hours, obtains the Bacillus acidi lactici shake-flask seed on the triangular flask fluid nutrient medium; Described culture medium is the mixed liquor in following ratio preparation: soy peptone 1%, glucose 1%, yeast soak powder 0.7wt%, potassium dihydrogen phosphate 0.5%, beef extract 0.5%, Triammonium citrate 1.5%, Tween-800.5%, sodium acetate 0.6%, pH=5.5～5.8,115 ℃, sterilized 15 minutes, for subsequent use;
3) production primary seed solution: first according to formulated first order seed liquid culture medium described later, through 121 ℃, sterilized 45 minutes, pH=5.5～5.8, be cooled to 37 ℃, then with step 2) the Bacillus acidi lactici shake-flask seed that obtains is with the inoculum concentration of 8v%, be inoculated in and produce in the used first class seed pot, cultivate 30h in 35 ℃, stir fast 150rpm, interval ventilation in 1 hour 5 minutes, throughput is 1.0vvm, until the Bacillus acidi lactici number reaches 1,000,000,000, obtain Bacillus acidi lactici and produce used primary seed solution; Described first order seed fermentation culture is the mixed liquor (W/V) in following ratio preparation: 120 order bean cake powders 0.5,120 order fish meal 0.5%, fish peptone 0.5%, lactose 0.2%, 120 order corn flour 0.5%, glucose 1%, NaCl 0.1%, Na2HPO
40.15%, synergist 0.05%, defoamer 0.03%, pH=5.5～5.8; Described synergist is open in first to file 200410008992.9, for by CaCl22～4g/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 culture medium is same as step 2) in culture medium;
4) the Bacillus acidi lactici first order seed that second order fermentation (producing fermentation): with step 3) obtains, ratio with 10% volume is inoculated in the fermentation tank that the production fermentation culture is housed, the same step 3) of the prescription of fermentation culture and controlled 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 Bacillus acidi lactici amount reaches greater than 109The CFU/ml zymotic fluid); shut down; obtain the zymotic fluid of Bacillus acidi lactici; at last according to dull and stereotyped used culture medium and the condition of culture of rejuvenation of cultivating of Bacillus acidi lactici; viable bacteria content to lactobacillus fermentation liquid carries out conventional plate count detection; according to count results, utilize the sterilization running water that viable bacteria content is diluted to respectively 108CFU/ml、
10
9CFU/ml and 1010CFU/ml obtains the reserve liquid (C) of lactobacillus fermentation liquid, treats post processing.
Embodiment 7, second order fermentation are cultivated saccharomyces cerevisiae (Saccharomyces cerevisiae) zymotic fluid (D)
1) dull and stereotypedly cultivates rejuvenation: the saccharomyces cerevisiae inclined-plane seed of preservation is adopted method streak inoculation on the saccharomyces cerevisiae plating medium of streak inoculation, cultivated 20～24 hours in 24～30 ℃, make the saccharomyces cerevisiae rejuvenation, and form single bacterium colony; The mixture of described culture medium 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 ℃, for subsequent use;
2) select typical single bacterium colony the saccharomyces cerevisiae culture plate of making shake-flask seed: from step 1) cultivating, be inoculated in the triangular flask liquid medium, in 28～32 ℃ of cultivations 24～37 hours, obtain the saccharomyces cerevisiae shake-flask seed liquid; The mixed liquor of described shake-flask seed nutrient solution for preparing by following ratio: 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 ℃, for subsequent use;
3) production primary seed solution: first according to saccharomyces cerevisiae culture medium prescription preparation first order seed liquid culture medium described later, through 121 ℃, sterilized 45 minutes, pH=5.8～6.0 are cooled to 35 ℃, then with step 2) the 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 ℃, mixing speed 180rpm, throughput is 1.5VVM, obtains saccharomyces cerevisiae and produces used primary seed solution; Described saccharomyces cerevisiae one-level kind nutrient solution is the mixed liquor in following ratio preparation: 120 order bean cake powders 0.9%, 120 order fish meal 0.8%, 120 order corn flours 0.9%, glucose 0.8%, NaCl0.2%, Na2HPO
40.5%, synergist 0.1%, defoamer 0.1%; Described synergist is open in first to file 200410008994.8, for by MgSO40.05～0.3g/L，CaCl
22.5～
4g/L，ZnSO
40.6～1.5g/L，FeSO
40.1～1.0g/L，MnSO
40.4～0.Sg/L，CuSO
40.02 the aqueous solution that～0.08g/L forms.
4) the saccharomyces cerevisiae first order seed that second order fermentation (producing fermentation): with step 3) obtains; ratio with 5% volume is inoculated in the fermentation tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and controlled condition; ferment after 36 hours; detect until the S. cervisiae number reaches 1,000,000,000/ml; shut down; obtain the zymotic fluid of S. cervisiae; at last according to dull and stereotyped used culture medium and the condition of culture 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 running water that viable bacteria content is diluted to respectively 108CFU/ml、10
9CFU/ml and 1010CFU/ml obtains the reserve liquid (D) of yeast fermentation broth, treats post processing.
Embodiment 8, three grade fermemtation are cultivated saccharomyces cerevisiae (Saccharomyces cerevisiae) zymotic fluid (D)
1) dull and stereotypedly cultivates rejuvenation: the saccharomyces cerevisiae inclined-plane seed of preservation is adopted method streak inoculation on the saccharomyces cerevisiae plating medium of streak inoculation, cultivated 20～24 hours in 24～30 ℃, make the saccharomyces cerevisiae rejuvenation, and form single bacterium colony; The mixture of described culture medium 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 ℃, for subsequent use;
2) select typical single bacterium colony the saccharomyces cerevisiae culture plate of making shake-flask seed: from step 1) cultivating, be inoculated in the triangular flask liquid medium, in 28～32 ℃ of cultivations 24～37 hours, obtain the saccharomyces cerevisiae shake-flask seed liquid; The mixed liquor of described shake-flask seed nutrient solution for preparing by following ratio: 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 ℃, for subsequent use;
3) production primary seed solution: first according to saccharomyces cerevisiae culture medium prescription preparation first order seed liquid culture medium described later, through 121 ℃, sterilized 45 minutes, pH=5.8～6.0 are cooled to 32～37 ℃, then with step 2) the saccharomyces cerevisiae shake-flask seed that obtains is by the inoculum concentration of 8v%, be inoculated in the first class seed pot, in 32 ℃ of cultivations 35 hours, stir fast 220rpm, throughput is 2.5VVM, obtains the saccharomyces cerevisiae primary seed solution; Described saccharomyces cerevisiae one-level kind nutrient solution is the mixed liquor in following ratio preparation: 120 order bean cake powders 0.9%, 120 order fish meal 0.8%, 120 order corn flours 0.9%, glucose 0.8%, NaCl0.2%, Na2HPO
40.5%, synergist 0.1%, defoamer 0.03%; Described synergist is open in first to file 200410008994.8, for by MgSO40.05～0.3g/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) the saccharomyces cerevisiae first order seed that second order fermentation (producing fermentation): with step 3) obtains, ratio with 7% volume is inoculated in the fermentation tank that the production fermentation culture is housed, the same step 3) of the prescription of fermentation culture and controlled condition, fermenting obtains the secondary seed of S. cervisiae after 30 hours.
5) the saccharomyces cerevisiae secondary seed that three grade fermemtation: with step 4) obtains; ratio with 5% volume is inoculated in the three grade fermemtation tank that the production fermentation culture is housed; the same step 3) of the prescription of fermentation culture and controlled condition; ferment after 36 hours; detect until the S. cervisiae number reaches 1,000,000,000/ml; shut down; obtain fermentation by saccharomyces cerevisiae liquid; at last according to dull and stereotyped used culture medium and the condition of culture 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 running water that viable bacteria content is diluted to respectively 108CFU/m1、
10
9CFU/ml and 1010CFU/ml obtains the reserve liquid (D) of yeast fermentation broth.
Liquor benefit-1～benefit-22 of embodiment 9～30, preparation additive for microbe feedstuff of the present invention
With the zymotic fluid (D) of the S. cervisiae of the zymotic fluid (C) of the Bacillus acidi lactici of the zymotic fluid (B) of the Bacillus cercus of the zymotic fluid (A) of the bacillus subtilis of embodiment 1～2, embodiment 3～4, embodiment 5～6 and embodiment 7～8 in the listed ratio preparation mixed liquor of table 1, obtain the liquor (Yishengkang) of additive for microbe feedstuff of the present invention, i.e. benefit-1～benefit-22.
The liquor of table 1, additive for microbe feedstuff of the present invention
| Somatotype | Numbering | Embodiment number | The zymotic fluid title | |||
| Bacillus subtilis A (v%) | Cured shape bacillus B (v%) | Bacillus acidi lactici C (v%) | 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 | ||
| Children's animal specific type in age | 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 ruminant 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 " refers to the code name of microbe additive name of product of the present invention " Yishengkang ".
Embodiment 31, preparation bacillus 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 CaCO350wt%, inferior powder 20wt%, corn protein powder 18% form, and the weight ratio of zymotic fluid and carrier is 0.5: 1, then 45 ℃ of dryings;
Then be crushed to more than 80 orders, obtain respectively bacillus subtilis dry powder, at last according to dull and stereotyped used culture medium and the condition of culture of rejuvenation of cultivating of bacillus, viable bacteria content for powder carries out conventional plate count detection, according to count results, utilize the carrier of present embodiment that viable bacteria content is diluted to respectively 108CFU/g、10
9CFU/g and 1010CFU/g obtains bacillus subtilis dry powder (E).
Embodiment 32, preparation bacillus 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) precipitated calcium carbonate, after mixing, press 115 ℃ of air intakes at spray dryer, 80 ℃ of air-out, 2kg, flow 1000ml/min, spray-drying are pressed in spray, then according to carrier and the Quality Control method of embodiment 31, viable bacteria content is diluted to respectively 108CFU/g、10
9CFU/g and 1010CFU/g obtains bacillus subtilis dry powder (F).
Embodiment 33, preparation Bacillus cercus dry powder (G)
The bacillus cereus fermentation liquid that embodiment 3 or 4 is obtained is added in the carrier that is pre-mixed, and described carrier is lightweight CaCO345wt%, inferior powder 30wt%, corn protein powder 20% form, and the weight ratio of zymotic fluid and carrier is 0.8: 1, then 45 ℃ of dryings;
Then be crushed to more than 80 orders, obtain respectively Bacillus cercus dry powder, at last according to dull and stereotyped used culture medium and the condition of culture of rejuvenation of cultivating of 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 respectively 108CFU/g、10
9CFU/g and 1010CFU/g, the Bacillus cercus dry powder (G) that obtains.
Embodiment 34, preparation Bacillus cercus dry powder (H)
The bacillus cereus fermentation liquid that embodiment 3 or 4 is obtained by weight volume ratio adds 0.5% dextrin and 3% precipitated calcium carbonate, after mixing, press 100 ℃ of air intakes at spray dryer, 60 ℃ of air-out, 2kg is pressed in spray, and flow 1100ml/min carries out spray-drying, then according to carrier and the Quality Control method of embodiment 33, viable bacteria content is diluted to respectively 108CFU/g、10
9CFU/g and 1010CFU/g obtains Bacillus cercus 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 concentrate.
Then add by skimmed milk power, white granulated sugar (sucrose), maltodextrin, gelatin in 1: 2: 1: the protective agent 15% (accounting for the weight/percent by volume of concentrate) that 1 ratio is mixed mixes with the lactic acid bacteria concentrate;
To add protectant lactic acid bacteria concentrate in-45 ℃ of freeze dryings; at last, according to dull and stereotyped used culture medium and the condition of culture of rejuvenation of cultivating of Bacillus acidi lactici, 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 respectively 108CFU/g、10
9CFU/g and 1010CFU/g obtains lactic acid bacteria freeze drying powder (I); Described carrier comprises: lightweight CaCO330wt%, inferior powder 15wt%, corn protein 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 concentrate.
This concentrate is added in the carrier that is pre-mixed, and described carrier is included as lightweight CaCO335wt%, inferior powder 15wt%, corn protein powder 20%, the weight ratio of concentrate and carrier is 0.5: 1, then fluidized bed drying, 45 ℃ of baking temperatures obtain dry powder of lactic acid bacteria, be crushed to more than 80 orders, at last, according to dull and stereotyped used culture medium and the condition of culture of rejuvenation of cultivating of Bacillus acidi lactici, 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 respectively 108CFU/g、10
9CFU/g and 1010CFU/g obtains dry powder of lactic acid bacteria (J).
Embodiment 37, preparation S. cervisiae dry powder (K)
The 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 concentrate.
This concentrate is added in the carrier that is pre-mixed, and described carrier is lightweight CaCO340wt%, inferior powder 20wt%, corn protein powder 20%, the weight ratio of concentrate and carrier is 0.7: 1, fluidized bed drying then, 50 ℃ of baking temperatures obtain S. cervisiae dry powder (K).
Pulvis benefit-23～benefit-33 of embodiment 38～47, preparation additive for microbe feedstuff 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 cercus dry powder (H) of the Bacillus cercus dry powder (G) of the bacillus subtilis dry powder (F) of the bacillus 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 additive for microbe feedstuff of the present invention, i.e. benefit-23～benefit-44.
The pulvis of table 2, additive for microbe feedstuff 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 (%) | Lactobacillus freeze-dried powder I (%) | The lactobacillus 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 | Children's animal specific in age | 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 | Ruminant is special-purpose | 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 " refers to the fluidized bed drying powder in the table; " spray powder " refers to spray dried powder. " benefit " refers to the code name of microbe additive name of product of the present invention " Yishengkang ".
Embodiment 51, additive for microbe feedstuff of the present invention (Yishengkang, viable bacteria content 109/ ml or g) on the impact of the output of milk and incidence of mammits
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 the feeding cow basal diet, feed per ton adds 1kg benefit-1 additive for microbe feedstuff;
The C group is added the Yishengkang group, and except the feeding cow basal diet, feed per ton adds 1kg benefit-5 additive for microbe feedstuff;
The D group is added the Yishengkang group, and except the feeding cow basal diet, feed per ton adds 1kg benefit-7 additive for microbe feedstuff;
The E group is added the Yishengkang group, and except the feeding cow basal diet, feed per ton adds 1kg benefit-18 additive for microbe feedstuff;
The F group is added the Yishengkang group, and except the feeding cow basal diet, feed per ton adds 1kg benefit-40 additive for microbe feedstuff;
Continuous 30 days, and press above-mentioned grouping repeated test 4 times, then record each group its output of milk of each detection and incidence of mammits are analyzed, and list in table 3.
The milk cow basal diet, prescription (percentage by weight) corn 45%, wheat bran 10%, rape cake 9%, cotton dregs 10.0%, dry beer vinasse 5.0%, Soybean Meal 18.0%, stone flour 1.0%, salt 1.0%, premix 1%.
Milk production of cow and incidence of mammits before and after table 3, the test
| Group/performance | Tested front 15 days | Tested rear 15 days | ||
| The per day output of milk (kg) | Incidence of mammits (%) | The average output of milk (kg) | Incidence of mammits (%) | |
| 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 |
As can be seen from Table 3, Yishengkang can significantly improve the per day output of milk of milk cow, and obviously reduce the incidence of mammits 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 service time to have cumulative effect less than early stage. To sum up test shows that Yishengkang can significantly improve the per day output of milk of milk cow, and can reduce the incidence of mammits of milk cow, and main cause is because the Yishengkang microbe additive can improve due to the immunity of milk cow.
Embodiment 52, additive for microbe feedstuff of the present invention (Yishengkang, viable bacteria content 108/ ml or g) on the impact of performance in layers, Egg Quality, pouity dwelling place environment
Share the identical later stage sea of laying eggs of age in days and match 15000 of brown shell layers, divide 5 groups, one group 3000 of every buildings, minute 3 repetitions, 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 additive for microbe feedstuff in laying hens essential ration;
The C group is added the Yishengkang group, adds 1kg benefit-23 additive for microbe feedstuff in laying hens essential ration;
The D group is added the Yishengkang group, adds 1kg benefit-27 additive for microbe feedstuff in laying hens essential ration;
The E group is added the Yishengkang group, adds 1kg benefit-38 additive for microbe feedstuff in laying hens essential ration;
The laying hens essential ration prescription (percentage by weight) 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%, vegetable oil 2.0%, 1% premix 1.0%.
Front 7 days is preliminary trial period, and rear 23 days is experimental period, and presses above-mentioned grouping repeated test 3 times, has measured NH in the house3、H
2The indexs such as S concentration, broken rate of eggshell, albumen quality (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, the indices of egg illustrate that all significantly better than control group this activated peptide as additive has played good facilitation in the production performance that improves laying hen and Egg Quality. Because Yishengkang can be regulated the gut flora balance, promote the secretory volume of microbial enzyme, improve digestive enzyme activity, and then the absorption of raising feed nitrogen nutrition element transforms and utilization ratio, indirectly reduce the content of ammonia and hydrogen sulfide in the pouity dwelling place, good economy and ecological benefits are arranged.
Embodiment 53～56, additive for microbe feedstuff of the present invention (Yishengkang) are on the impact of piglet, growing and fattening pigs, broiler chicken and grass carp
Table 5,
| Embodiment number | Yishengkang | Test method | Experimental scale | Result of the test |
| 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 aureomycin 100g/t contrast, and B, C, D, E, F, G group are 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 aureomycin 100g/t, sulfanilamide (SN) 100g/t contrast, and B, C, D, E, F group are 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 feed intake of fattening pig, daily gain; Feed efficiency improves, and diarrhea rate and death rate reduce, and every production performance is not less than adds antibiotic colony; The pig house stink reduces, and dressing percentage is similar or higher to interpolation antibiotic colony with lean meat percentage, 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 | Large chicken is added: benefit-28 benefit-29 benefit-30 benefit-31 benefits-38 (contain bacterium 1010) contrast adds terramycin 150g/t chicken and add among the 1.5kg/t chicken and add the large chicken of 150kg/t and add 20g/t | 24000 of broiler chicken | Increase the deposition of chicken body internal protein and reduce abdomen fat rate (P＜0.05), obviously reduce pH value (p＜0.05 of duodenum, jejunum and ileum,), significantly suppress the E.Coli breeding in (minimizing) jejunum and the rectum, and can improve the content (p＜0.05) of Bacillus acidi lactici in the enteron aisle, extremely be conducive to the health of chicken and reduce stink. |
| Benefit-25 benefit-26 benefit-36 benefits-37 (contain bacterium 109) | Feed granulating, every group of 4000 broiler chicken, 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 contrast group improves 6%, and daily gain contrast group improves 12.3%, and survival rate improves 20.7%, and the regularity of test group is good, and resistance against diseases is strong. Water quality control aspect, 2.8 meters, 3 mu inputs of depth of water Yishengkang liquid 1kg. Ammoniacal nitrogen (NH as a result3-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 mossambica reduces 61.3% simultaneously. |
As can be seen from Table 5, the Yishengkang additive provided by the invention of feeding, the indices of piglet, the difference raising stage of breeding pig, meat chick and grass carp is all significantly better than control group (interpolation antibiotic), 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 livestock water quality, improve the effects such as animal product quality, good economy and ecological benefits are arranged.
Claims (11)
1, a kind of additive for microbe feedstuff, its universal liquor is that the zymotic fluid of bacillus subtilis, Bacillus cercus, Bacillus acidi lactici and S. cervisiae difference fermented and cultured is cultivated used culture medium and the condition of culture 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 respectively 108CFU/ml、10
9CFU/ml or 1010CFU/ml, then formulated according to following volume ratio:
Fermentation of bacillus subtilis liquid 10～30v%; Bacillus cereus fermentation liquid 10～30v%;
Lactobacillus fermentation liquid 30～50v%; Saccharomyces cerevisiae fermented liquid 10～30v%
The zymotic fluid of described bacillus subtilis and Bacillus cercus is to be obtained by liquid deep layer fermenting, comprises following step:
1) dull and stereotypedly cultivates rejuvenation: bacillus subtilis and cured shape bacillus bacterial classification are adopted respectively method streak inoculation on the bacillus plating medium of streak inoculation, cultivated 12～24 hours in 25～35 ℃, make respectively bacillus subtilis and the rejuvenation of cured shape bacillus, and form single bacterium colony; Described bacillus plating medium is the mixture by following w/v preparation: peptone 0.5～1%, glucose 0.5～2%, beef extract 0.5～1.5%, potassium dihydrogen phosphate 0.3～0.6%, agar 3～4%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, for subsequent use;
2) choose respectively single bacterium colony the bacillus subtilis of making shake-flask seed: from step 1) cultivating and the cured shape bacillus bacterium colony, be inoculated in the bacillus liquid culture medium, in 28～37 ℃ of cultivations 20～30 hours, obtain bacillus subtilis and cured shape bacillus shake-flask seed liquid; Described bacillus liquid culture medium is the mixed liquor by following w/v preparation: peptone 0.5～1%, glucose 0.5～2%, beef extract 0.5～1.5%, potassium dihydrogen phosphate 0.3～0.6%, 3.08% manganese sulfate solution 0.5～1.5%, pH=7.5～7.6, sterilized 30 minutes for 121 ℃, for subsequent use;
3) the bacillus subtilis shake-flask seed liquid that production primary seed solution: with step 2) obtains and cured shape bacillus shake-flask seed liquid are with the inoculum concentration of 2～5v%, be inoculated in respectively in the first order seed liquid culture medium of producing used first class seed pot, cultivate 18～24h in 32～37 ℃, mixing speed 150～200rpm, throughput is 1～2vols/vol/min, obtains respectively the primary seed solution of bacillus subtilis and Bacillus cercus; Described first order seed liquid culture medium prescription is the mixed liquor by following w/v preparation: 120 order bean cake powders 0.5～2.0%, 120 order fish meal 0.2～1.5%, 120 order corn flours 0.5～1.0%, glucose 1.0～4.5%, NaCl 0.1～0.3%, NaOH 0.05～0.10%, Na2HPO
40.1～0.5%, synergist 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 synergist is by MgSO40.1～0.5g/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) bacillus subtilis that second order fermentation: with step 3) obtains and cured shape bacillus first order seed, be inoculated into 5～10% volume ratio respectively and be equipped with in the fermentation tank of producing fermentation culture, prescription and the controlled condition of fermentation culture are same as step 3) first order seed liquid culture medium, ferment after 16～24 hours, by microscopic examination, when being arranged, a large amount of sporulation stops fermentation;
The zymotic fluid of described Bacillus acidi lactici is to be obtained by liquid deep layer fermenting, comprises following step:
1) dull and stereotyped cultivation rejuvenation: with method streak inoculation on the Bacillus acidi lactici plating medium of Bacillus acidi lactici inclined-plane seed employing streak inoculation, in 33～38 ℃ of cultivations 24～48 hours, make the Bacillus acidi lactici rejuvenation, and form single bacterium colony; Described Bacillus acidi lactici plating medium is the mixture by following w/v preparation: soy peptone 0.5～1%, glucose 1～2%, yeast soak powder 0.3～0.7%, potassium dihydrogen phosphate 0.3～0.5%, beef extract 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 ℃, for subsequent use;
2) choose single bacterium colony in the Bacillus acidi lactici bacterium colony of making shake-flask seed: with step 1) cultivating, be inoculated in the Bacillus acidi lactici fluid nutrient medium, the employing conventional method in 35～40 ℃ of cultivations 24～48 hours, obtains the Bacillus acidi lactici shake-flask seed on the triangular flask fluid nutrient medium; Described culture medium is the mixed liquor by following w/v preparation: soy peptone 0.5～1%, glucose 1～2%, yeast soak powder 0.3～0.7%, potassium dihydrogen phosphate 0.3～0.5%, beef extract 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 ℃, for subsequent use;
3) the Bacillus acidi lactici shake-flask seed that production primary seed solution: with step 2) obtains is with the inoculum concentration of 6～12v%, be inoculated in the first order seed liquid culture medium of producing used first class seed pot, cultivate 17～60h in 35～38 ℃, mixing speed 150～200rpm, interval ventilation in 1 hour 5～10 minutes, throughput is 1～2.5vols/vol/min, until the Bacillus acidi lactici number reaches 1,000,000,000, obtains the primary seed solution of Bacillus acidi lactici; Described first order seed liquid culture medium prescription is the mixed liquor by following w/v preparation: 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 corn flours 0.5～1.0%, glucose 1～2.5%, NaCl 0.1～0.3%, Na2HPO
40.15～0.3%, synergist 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 synergist is by CaCl2
2～4g/L，ZnSO
4 0.5～1.5g/L，FeSO
4 0.2～1.0g/L，CuSO
4 0.01～0.08g/L，Co(NO
3)
2
0.02～0.06g/L，MgSO
40.1 the aqueous solution that～0.5g/L forms;
4) the Bacillus acidi lactici first order seed that second order fermentation: with step 3) obtains, be inoculated in the fermentation tank that the production fermentation culture is housed with 5～10% volume ratios, prescription and the controlled condition of fermentation culture are same as step 3) first order seed liquid culture medium, ferment after 17～60 hours, detection pH value drops at 5.0 o'clock and stops fermentation;
The zymotic fluid of described saccharomyces cerevisiae is to be obtained by liquid deep layer fermenting, comprises following step:
1) dull and stereotyped cultivation rejuvenation: with method streak inoculation on the saccharomyces cerevisiae plating medium of saccharomyces cerevisiae inclined-plane seed employing streak inoculation, in 24～30 ℃ of cultivations 20～24 hours, make the saccharomyces cerevisiae rejuvenation, and form single bacterium colony; Described saccharomyces cerevisiae plating medium is the mixture by following 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 ℃, for subsequent use;
2) choose single bacterium colony the saccharomyces cerevisiae culture plate of making shake-flask seed: from step 1) cultivating, be inoculated in the triangular flask liquid medium, in 28～32 ℃ of cultivations 24～37 hours, obtain the saccharomyces cerevisiae shake-flask seed liquid; Described nutrient solution is the mixed liquor by following 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%, and pH=5.8～6.0,121 ℃ sterilization 15 minutes is for subsequent use;
3) the saccharomyces cerevisiae shake-flask seed that production primary seed solution: with step 2) obtains is by the inoculum concentration of 2～12v%, be inoculated in the first order seed nutrient solution of first class seed pot, cultivated 24～37 hours in 28～32 ℃, mixing speed 160～220rpm, throughput is 1.0～3.0vols/vol/min, obtains the primary seed solution of saccharomyces cerevisiae; Described saccharomyces cerevisiae first order seed nutrient solution is the mixed liquor by following w/v preparation: 120 order bean cake powders 0.5～1.5%, 120 order fish meal 0.2～0.8%, 120 order corn flours 0.5～1.0%, glucose 0.3～1.0%, NaCl 0.1～0.3%, Na2HPO
40.2～0.5%, synergist 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 synergist is by MgSO40.05～0.3g/L，CaCl
2
2.5～4g/L，ZnSO
4 0.6～1.5g/L，FeSO
4 0.1～1.0g/L，MnSO
4 0.4～0.8g/L，CuSO
40.02 the aqueous solution that～0.08g/L forms;
4) the saccharomyces cerevisiae first order seed that second order fermentation: with step 3) obtains, be inoculated in the fermentation tank that the production fermentation culture is housed with 4～10% volume ratios, prescription and the controlled condition of fermentation culture are same as step 3) the first order seed nutrient solution, ferment after 24～60 hours, detection S. cervisiae number stops fermentation when reaching 1,000,000,000/ml.
2, a kind of additive for microbe feedstuff, its children's animal specific type liquor in age is that the fermentation of bacillus liquid described in the claim 1, lactobacillus fermentation liquid, yeast fermentation broth are cultivated used culture medium and the condition of culture 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 respectively 108CFU/ml、10
9CFU/ml or 1010CFU/ml, then formulated according to following volume ratio:
Fermentation of bacillus subtilis liquid 10～20v%; Bacillus cereus fermentation liquid 10～20v%;
Lactobacillus fermentation liquid 40～60v%; Saccharomyces cerevisiae fermented liquid 15～25v%.
3, a kind of additive for microbe feedstuff, its above the average age for marriage animal specific type liquor is that the fermentation of bacillus liquid described in the claim 1, lactobacillus fermentation liquid, yeast fermentation broth are cultivated used culture medium and the condition of culture 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 respectively 108CFU/ml、10
9CFU/ml or 1010CFU/ml, then formulated according to following volume ratio:
Fermentation of bacillus subtilis liquid 20～40v%; Bacillus cereus fermentation liquid 15～30v%;
Lactobacillus fermentation liquid 20～40v%; Saccharomyces cerevisiae fermented liquid 10～25v%.
4, a kind of additive for microbe feedstuff, its ruminant tailored version liquor is that fermentation of bacillus liquid claimed in claim 1, lactobacillus fermentation liquid, yeast fermentation broth are cultivated used culture medium and the condition of culture 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 respectively 108CFU/ml、10
9CFU/ml or 1010CFU/ml, then formulated according to following volume ratio:
Fermentation of bacillus subtilis liquid 15～30v%; Bacillus cereus fermentation liquid 10～30v%;
Lactobacillus fermentation liquid 30～40v%; Saccharomyces cerevisiae fermented liquid 15～35v%.
5, a kind of additive for microbe feedstuff, its granulation feed tailored version liquor is that fermentation of bacillus liquid claimed in claim 1, lactobacillus fermentation liquid, yeast fermentation broth are cultivated used culture medium and the condition of culture 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 respectively 108CFU/ml、10
9CFU/ml or 1010CFU/ml, then formulated according to following volume ratio:
Fermentation of bacillus subtilis liquid 30～40v%; Bacillus cereus fermentation liquid 20～30v%;
Lactobacillus fermentation liquid 20～30v%; Saccharomyces cerevisiae fermented liquid 10～20v%.
6, a kind of additive for microbe feedstuff, its universal pulvis for bacillus subtilis, Bacillus cercus, Bacillus acidi lactici and S. cervisiae respectively the dry powder that makes of the zymotic fluid of fermented and cultured cultivate used culture medium and the condition of culture 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 respectively 108CFU/g、10
9CFU/g or 1010CFU/g, described carrier proportioning is: lightweight CaCO3Then 25～50wt%, inferior powder 20～30wt%, corn protein powder 15～20wt% form than preparation according to following weight:
Bacillus subtilis dry powder 10～30wt%; Bacillus cercus dry powder 10～30wt%;
Lactobacillus dry powder 30～50wt%; S. cervisiae dry powder 10～30wt%;
The dry powder of described bacillus subtilis and Bacillus cercus is made by following method, specifically comprises following step:
With the zymotic fluid of the bacillus subtilis described in the claim 1 and the zymotic fluid of Bacillus cercus, weight ratio according to zymotic fluid 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 respectively bacillus subtilis dry powder and Bacillus cercus dry powder; Described carrier proportioning is: lightweight CaCO325～50wt%, inferior powder 20～30wt%, corn protein powder 15～20wt%;
Or the zymotic fluid of the zymotic fluid of bacillus subtilis and Bacillus cercus by volume added the dextrin of fermentating liquid volume 0.3～0.8% and 3～10% precipitated calcium carbonate with weight ratio respectively, after mixing, press 100～130 ℃ of air intakes at spray dryer, 60～90 ℃ of air-out, 2kg is pressed in spray, flow 1000～1500ml/min carries out spray-drying, obtains bacillus subtilis spraying dry powder and Bacillus cercus spraying dry powder;
Described dry powder of lactic acid bacteria is made by following method, specifically comprises following step:
1) zymotic fluid of the lactic acid bacteria described in the claim 1 is concentrated to 40～60% of original volume being lower than under 45 ℃, negative-pressure vacuum condition;
2) adding accounts for the protective agent of the weight/percent by volume 15～20% of concentrate, in-25～-45 ℃ of freeze dryings, obtains the lactic acid bacteria freeze drying powder after mixing; Described protective agent be skimmed milk power, white granulated sugar, maltodextrin, gelatin in 1: 2: 1: 1 ratio mixes;
Or the concentrate of lactic acid bacteria added in the carrier that is pre-mixed, the weight ratio of concentrate and carrier is 0.4～0.9: 1, then through 45～60 ℃ of dryings of fluid bed, is crushed to more than 80 orders, obtains dry powder of lactic acid bacteria; Described carrier comprises: lightweight CaCO330～40wt%, inferior powder 15～30wt%, corn protein powder 18～20wt%;
Described S. cervisiae dry powder is made by following method, specifically comprises following step:
1) zymotic fluid of the S. cervisiae described in the claim 1 is concentrated to 40～60% of original volume being lower than under 45 ℃, the condition of negative-pressure vacuum, obtains the concentrate of S. cervisiae;
2) concentrate of S. cervisiae is added in the carrier that is pre-mixed, the weight ratio of concentrate and carrier is 0.6～0.9: 1, fluidized bed drying then, and 45～60 ℃ of baking temperatures obtain S. cervisiae dry powder; Described carrier comprises: lightweight CaCO330～50wt%, inferior powder 20～30wt%, corn protein powder 18～20wt%.
7, a kind of additive for microbe feedstuff, its children's animal specific type pulvis in age is that bacillus dry powder claimed in claim 6, lactobacillus dry powder are cultivated used culture medium and the condition of culture 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 respectively 108CFU/g、10
9CFU/g or 1010CFU/g, described carrier proportioning is: lightweight CaCO3Then 25～50wt%, inferior powder 20～30wt%, corn protein powder 15～20wt% form than preparation according to following weight:
Bacillus subtilis dry powder 10～20wt%; Bacillus cercus dry powder 10～20wt%;
Lactobacillus dry powder 40～60wt%; S. cervisiae dry powder 15～25wt%.
8, a kind of additive for microbe feedstuff, its above the average age for marriage animal specific type pulvis is that bacillus dry powder claimed in claim 6, lactobacillus dry powder are cultivated used culture medium and the condition of culture 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 respectively 108CFU/g、10
9CFU/g or 1010CFU/g, described carrier proportioning is: lightweight CaCO3Then 25～50wt%, inferior powder 20～30wt%, corn protein powder 15～20wt% form than preparation according to following weight:
Bacillus subtilis dry powder 20～40wt%; Bacillus cercus dry powder 15～30wt%;
Lactobacillus dry powder 20～40wt%; S. cervisiae dry powder 10～25wt%.
9, a kind of additive for microbe feedstuff, its ruminant tailored version pulvis is that bacillus dry powder claimed in claim 6, lactobacillus dry powder are cultivated used culture medium and the condition of culture 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 respectively 108CFU/g、10
9CFU/g or 1010CFU/g, described carrier proportioning is: lightweight CaCO3Then 25～50wt%, inferior powder 20～30wt%, corn protein powder 15～20wt% form than preparation according to following weight:
Bacillus subtilis dry powder 15～30wt%; Bacillus cercus dry powder 10～30wt%;
Lactobacillus dry powder 30～40wt%; S. cervisiae dry powder 15～35wt%.
10, a kind of additive for microbe feedstuff, its granulation feed tailored version pulvis is that bacillus dry powder claimed in claim 6, lactobacillus dry powder are cultivated used culture medium and the condition of culture 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 respectively 108CFU/g、10
9CFU/g or 1010CFU/g, described carrier proportioning is: lightweight CaCO3Then 25～50wt%, inferior powder 20～30wt%, corn protein powder 15～20wt% form than preparation according to following weight:
Bacillus subtilis dry powder 30～40wt%; Bacillus cercus dry powder 20～30wt%;
Lactobacillus dry powder 20～30wt%; S. cervisiae dry powder 10～20wt%.
11, the described additive for microbe feedstuff of one of a kind of claim 1 to 10 is as the purposes of producing the enzyme probiotic.
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| CN103918915A (en) * | 2014-04-21 | 2014-07-16 | 吉林农业大学 | Compound microorganism additive for preserving total mixed ratio of herbivores and application method thereof |
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