CN1520739A - Bacilli fungi mixed fermentation for producing microbial formulation and compound enzyme feed additive - Google Patents
Bacilli fungi mixed fermentation for producing microbial formulation and compound enzyme feed additive Download PDFInfo
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- CN1520739A CN1520739A CNA021048711A CN02104871A CN1520739A CN 1520739 A CN1520739 A CN 1520739A CN A021048711 A CNA021048711 A CN A021048711A CN 02104871 A CN02104871 A CN 02104871A CN 1520739 A CN1520739 A CN 1520739A
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Abstract
The present invention belongs to the field of biochemistry and microbiology. By means of mixed inoculation with Bacillus licheniformis, Bacillus cereus, Bacillus subtilis and aspergillus and thick-layer ventilating fermentation in solid fermenting machine, composite feed additive with high quality ecological preparation and composite enzyme of several kinds of enzyme and high activity may be obtained. The product is suitable for farm animal, aquatic product and other fed animal and may be used to raise feed utilization, raise production performance and lower raising cost.
Description
The invention belongs to biological chemistry and microbiology field.
At present, feed resource is in short supply day by day, and the herding environmental pollution is on the rise, and how fully to improve the utilization ratio of animal to feed, reduces the waste of feedstuff raw material, and to reduce herding production be one of problem anxious to be solved to the pollution of environment.Secondly, because the extensive life-time service of antibiotic etc fodder additives causes the pathogenic bacterium of people is developed immunity to drugs, reduced or forbidden in feed, using microbiotic as growth stimulant in developed country.Therefore safe and effective fodder additives is sought in an urgent demand, and the antibiotic as an alternative green feed additive of fodder enzyme preparation and probiotics is subjected to common concern.
Fodder enzyme preparation is to use modern biotechnology, select for use peculiar microorganism to produce biochemicals through fermentation with catalytic activity, it is to improving food conversion ratio, widen the feedstuff raw material source, improve breeding performonce fo animals, reduce organism and noxious gas emission, economic benefit and obvious social benefit, the effect of fodder enzyme preparation and result of use have obtained the generally approval of Animal nutrition educational circles and aquaculture, and therefore, fodder enzyme preparation is very fast in the China recent years development.
Along with of propagation and the application of little ecological theory in herding circle, Animal nutrition educational circles has carried out the applied research of a large amount of little ecological products, people are on the basis of formula nutritional, and the applied ecology principle is adjusted in the animal body and microbe species and quantity in the environment, keep microecological balance and body health in the body, excite the ability of digesting and assimilating, strengthen body's immunity, reach disease resistance, the effect that improves production performance is obvious, thereby probiotics uses universal day by day.
But fodder enzyme preparation and poultry are to produce respectively with probiotics at present, carry out composite use then.Purpose of the present invention is exactly the method that original zymin and probiotics are produced respectively to be changed into disposablely produce simultaneously, promptly obtains existing high-quality compound enzymic preparation, has the method for composite prod of the probiotics of high-quality amount again.
Core content of the present invention and characteristics:
1. increased the production bacterial classification of the probiotics of three strain strong stress resistances on patent of invention " production of aspergillus niger one time fermentation obtains the method for the complex enzyme of several kinds of enzyme and with high enzyme activity " basis of our company: the 1-3 kind mixed fermentation in Bacillus licheniformis, bacillus cereus and the subtilis is produced.Three kinds of bacteriums of the present invention: Bacillus licheniformis (Bacillus licheniformisHQ0101), bacillus cereus (Bacillus cereus HQ0102), subtilis (Bacillus subtilis HQ0103)) and a kind of fungi aspergillus niger (Aspergillus nigerHQ978).Above-mentioned four kinds of microorganisms are all in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation.Its address is the BeiJing ZhongGuanCun, and preservation date is on January 23rd, 2002, and preserving number is respectively CGMCC NO:
0697,
0698,
0699,
0696
2. probiotics and compound enzymic preparation mixed fermentation are produced, and have reduced wherein a kind of production process, have reduced production cost, and improve usage ratio of equipment;
3. constant product quality, good, existing high-quality probiotics (viable count 〉=8 * 10
10) have high-quality prozyme again, (aspartic protease 10000～11000u/g, cellulase 18000～20000u/g, zytase 60000～80000u/g, beta-glucanase 40000～60000u/g, polygalacturonase 18000～20000u/g new composite fodder additives, this product is suitable for poultry, fowl, aquatic animal use, and effect is remarkable.
Concrete implementation step is as follows:
One, strain preparation technology
(1) bacteria culture preparation: be divided into the three-class strain preparation
1. first class inoculum: first class inoculum is expanded numerous by the most original slant strains, and slant strains can expand and meets 40-50 and prop up first class inoculum, and substratum is a beef-protein medium, and pH value is 7.0-7.2, and culture temperature is 35-36 ℃, and incubation time is 48 hours.
2. second class inoculum: substratum, bloom 25%, dregs of beans 25%, rice bran 29%, Semen Maydis powder 20%, sucrose 1%, PH7.2-7.4, the bottled 10g of each 300ml glass, material water is 1: 0.8,121 ℃ of sterilizations, cooling inserts the outstanding 5ml of first class inoculum spore, stirs evenly, and cultivates 48 hours for 35-36 ℃.
3. the three-class strain substratum is formed same second class inoculum.Method is with our company's patent of invention " simple container of microbial strains enlarged culturing ", (22 * 30cm) adorn 90 gram wet feeds to each plastics bag, 121 ℃ of sky 30min, cooling, insert the second class inoculum spore suspension (this suspension for every secondary triangular flask seed bottle add water 300ml wash get) 25ml, shake up 34-35 ℃, cultivated 48-50 hour, and used for fermentative production.
(2), aspergillus niger strain preparation technology is with " production of aspergillus niger one time fermentation obtains the complex enzyme of several kinds of enzyme and with high enzyme activity method." patent of invention, promptly be divided into one, two, three strain preparation.
(1) the first class inoculum first class inoculum is expanded numerously by the most original slant strains, and slant strains can expand and meets 30-40 and prop up first class inoculum.The substratum base is PDA, natural pH value, culture temperature 28-30 ℃, incubation time 72 hours.
(2) second class inoculum substratum base is a wheat bran, each 300ml triangular flask, and dress 10g wheat bran, material: water is 1: 0.6,121 ℃ of sterilization 30min cool off＜40 ℃ and meet first class inoculum spore suspension 5ml, cultivate 72-74 hour for 28-30 ℃.
(3) the three-class strain substratum is wheat bran dregs of beans (8: 2), mixing, material: water is 1: 0.7, again mixing, the plastics bag (22 * 30cm) of packing into, every packed 90 gram batch mixings, 121 ℃ of sterilization 30min, cooling, insert secondary bacterium spore suspension 15ml, (this spore suspension for each secondary bacterium triangular flask add 300ml spore liquid is washed get) tremble and pinch evenly, cultivated 72-74 hour, and used for 28 ℃-30 ℃ for fermentative production.
Two, fermentative production
1. the fermentation machine adopts solid ventilating fermentation machine; (butt meter) 1000kg feeds intake at every turn.
2. fermention medium preparation substratum is a wheat bran 65～75%, bloom 10～15%, dregs of beans 13～15%, ammonium sulfate 3～4%, potassium primary phosphate 1～2%, natural PH, earlier wheat bran, dregs of beans, rice bran are stirred, ammonium sulfate and potassium primary phosphate are dissolved in the tap water that is equivalent to siccative 70% and spray in the material, stir autoclaving 40-50min.Cool off standby.
3. inoculation evenly sprays into preceding paragraph cooling and fermentation substratum with the three-class strain of the sterilized water washing bacterium that is equivalent to fermention medium siccative 50% and the spore (bacterium: fungi is 1: 1) of aspergillus niger three-class strain.
4. the material input that will connect bacterial classification of fermenting is cultivated through the fermentor tank of sterilization.Design temperature 30-31 ℃, generally cultivate 20-24h, thalli growth well lumps, and carries out the stirring first time, carries out first and second time stirring with backsight thalli growth caking situation, is advisable with the agglomerate that keeps 3-5cm.When first and second time stirring, if culture material moisture＜30% o'clock can take the circumstances into consideration to spray into sterilized water, be beneficial to thalli growth and produce enzyme, fermentation time generally is controlled at 55-60h and finishes fermentation.
Three, oven dry and pulverizing
After going out jar, bent material is through crusher in crushing, oven drying at low temperature, general＜45 ℃ dry and contain moisture 8-10% to material and get final product, pulverize then, measure the qualified finished product that gets.The viable count of product 〉=8 * 10
10); Enzyme and enzyme are lived; Aspartic protease 10000～11000u/g, cellulase 18000～20000u/g, zytase 60000～80000u/g, beta-glucanase 40000～60000u/g, polygalacturonase 18000～20000u/g.
Claims (6)
1. four kinds of used microorganisms of the present invention, it is characterized in that three kinds of bacteriums: Bacillus licheniformis (Bacillus licheniformis), bacillus cereus (Bacillus cereus), subtilis (Bacillus subtilis) and a kind of fungi aspergillus niger (Aspergillusniger), they are numbered HQ0101, HQ0102, HQ0103 and HQ978 respectively.Preserving number is CGMCC NO:
0697,
0698,
0699,
0696
2. according to the described three kinds of bacteriums of claim 1 (1-3 kind) and a kind of fungi combined inoculation, adopt the production of solid fermentation machine, one time fermentation can obtain the high-quality probiotics and the novel complex enzyme feed additive of prozyme;
3. according to the described product of claim 2, it is characterized in that: (1) high-content probiotics (viable count 〉=8 * 10
10); (2) high vigor prozyme (aspartic protease 8000～10000u/g, zytase 60000～80000u/g, cellulase 18000～20000u/g, beta-glucanase 40000～60000u/g, tartaric acid enzyme 18000～20000u/g), NEW TYPE OF COMPOSITE fodder additives.This product is suitable for poultry, fowl, aquatic products etc. and propagates animal artificially;
4. according to the production method of the described product of claim 3, it is characterized in that it comprises strain preparation technology and zymotechnique;
5. be the production of hybrid seeds respectively of various bacterial classifications according to the described strain preparation technology characteristics of claim 4, and be divided into one, two, three bacterial classification, step by step the technology of amplification culture bacterial classification:
(I) bacteria culture preparation: be divided into the three-class strain preparation
(1) first class inoculum: first class inoculum is expanded numerous by the most original slant strains, and slant strains can expand and meets 40-50 and prop up first class inoculum, and substratum is a beef-protein medium, and pH value is 7.0-7.2, and culture temperature is 35-36 ℃, and incubation time is 48 hours.
(2) second class inoculum: substratum, bloom 25%, dregs of beans 25%, rice bran 29%, Semen Maydis powder 20%, sucrose 1%, PH7.2-7.4, the bottled 10g of each 300ml glass, material water is 1: 0.8,121 ℃ of sterilizations, cooling inserts first class inoculum, and every inclined-plane connects three bottles, stirs evenly, and cultivates 48 hours for 35-36 ℃.
(3) the three-class strain substratum is formed same second class inoculum.Method is with our company's patent of invention " the microbial strains enlarged culturing is sent out simple container ", each plastics bag (22 * 30cm) bag of 90 gram wet feed, 121 ℃ of sky 30min, cooling, insert the second class inoculum spore suspension (this suspension for every secondary triangular flask seed bottle add water 300ml wash get) 25ml, shake up 34-35 ℃, cultivated 48-50 hour, and used for fermentative production.
(II), aspergillus niger strain preparation technology is with " production of aspergillus niger one time fermentation obtains the complex enzyme of several kinds of enzyme and with high enzyme activity method." patent of invention, promptly be divided into one, two, three strain preparation.
(1) the first class inoculum first class inoculum is expanded numerously by the most original slant strains, and slant strains can expand and meets 30-40 and prop up first class inoculum.The substratum base is PDA, 28-30 ℃ of natural pH value culture temperature, incubation time 72 hours.
(2) second class inoculum substratum base is a wheat bran, each 300ml triangular flask, and dress 10g wheat bran, material: water is 1: 0.6,121 ℃ of sterilization 30min cool off＜40 ℃ and meet first class inoculum spore suspension 5ml, cultivate 72-74 hour for 28-30 ℃.
(3) the three-class strain substratum is wheat bran dregs of beans (8: 2), mixing, material: water is 1: 0.7, again mixing, the plastics bag (22 * 30cm) of packing into, every packed 90 gram batch mixings, 121 ℃ of sterilization 30min, cooling, insert secondary bacterium spore suspension 15ml, (this spore suspension for each secondary bacterium triangular flask add 300ml spore liquid is washed get) tremble and pinch evenly, cultivated 72-74 hour, and used for 28 ℃-30 ℃ for fermentative production.
6. the feature according to the described zymotechnique of claim 4 is: fermention medium is wheat bran 65-75%, bloom 10-15%, dregs of beans 13-15%, ammonium sulfate 3-4%, potassium primary phosphate 1-2%, natural P
HProduce at solid fermentation machine thick-layer ventilating fermentation or shallow tray fermentation mode.Culture temperature 28-30 ℃, fermentation time 55-60h.
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