CN103173371A - Production of saccharomyces cerevisiae and lactobacillus acidophilus composite microbe preparation used for feed - Google Patents
Production of saccharomyces cerevisiae and lactobacillus acidophilus composite microbe preparation used for feed Download PDFInfo
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Abstract
The invention relates to a microbe preparation, and concretely relates to a production method of a saccharomyces cerevisiae and lactobacillus acidophilus composite microbe preparation used for feed, the production method is characterized in that concentrated saccharomyces cerevisiae mother liquor containing saccharomyces cerevisiae greater than or equal to 50 hundreds million/ml and the concentrated lactobacillus acidophilus mother liquor with high density containing lactobacillus acidophilus greater than or equal to 100 hundreds million/ml are mixed according to 1:1 with the volume ratio, and the composite microbe preparation product containing saccharomyces cerevisiae greater than or equal to 50 hundreds million/ml and containing lactobacillus acidophilus greater than or equal to 100 hundreds million/ml. The saccharomyces cerevisiae and lactobacillus acidophilus composite microbe preparation takes glucose, cane sugar, cane sugar honey and corn steep liquor as main fermentation raw materials, the fermental cultivation can be carried out at 30-34 DEG C, the product bacteria number is high, the quality is good, pH value is between 5.0-6.6, and the quality guarantee period is 18 monthes. The saccharomyces cerevisiae and lactobacillus acidophilus composite microbe preparation used for feed has simple production technology and good product application effect, can purify the culture environment, and can be used in the culture industries such as aquatic product and livestock and poultry.
Description
Background technology
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is also bread yeast or tell budding yeast, is and mankind's relation primary yeast the most widely.Be used for traditionally making bread and wine brewing, be used as the eucaryon model animals in modern molecule and cytobiology.The cellular form of yeast saccharomyces cerevisiae is spherical or ovoid, diameter 510 μ m.The mode of breeding is gemmation, and its carbon source nitrogenous source utilization is extensive, can utilize glucose, maltose, sucrose, semi-lactosi, pectinose, N.F,USP MANNITOL, succsinic acid, citric acid, wood sugar, sorbyl alcohol, fructose, ethanol and glycerine; Can not utilize rhamnosyl, starch, cellobiose, trehalose, ribitol, inositol, tetrahydroxybutane, melibiose.
Lactobacterium acidophilum (Lactobacillus acidophilus) belongs to lactobacillus, Gram-positive bacillus, and the end of bar is rounded, mainly exists in small intestine.Can secrete lactic acid, acetic acid and peptide antibiotic.Lactobacterium acidophilum is mainly used in adjusting the intestinal microflora balance, suppresses the propagation of enteron aisle undesirable microorganism, and pathogenic microorganism is had antagonistic action.Particularly to gastrointestinal dysfunction with take antibiotic and cause that the symptom such as gastrointestinal dysfunction has special efficacy, can make rapidly the flora in enteron aisle recover normal equilibrium, suppress the propagation of spoilage organism, have good health protection effect.
In former studies, the scientific worker all has comparatively deep research to the aspects such as taxonomy, physiological and biochemical property, fermentation condition and regulation and control thereof of yeast saccharomyces cerevisiae and Lactobacterium acidophilum.Applied research to yeast saccharomyces cerevisiae and Lactobacterium acidophilum also obtains very large progress.But to yeast saccharomyces cerevisiae and Lactobacterium acidophilum between the two interactive research and application report and few.Yeast saccharomyces cerevisiae and Lactobacterium acidophilum, both have common characteristics and are: growth and breeding speed is fast, easily cultivation, thalline and meta-bolites nutritious etc.So in recent years, its product of yeast saccharomyces cerevisiae and Lactobacterium acidophilum is widely used in all many-sides such as foodstuffs industry, fodder industry, aquaculture, environment protection and health care medicine; Therefore, more and more being subject to scientific worker and business people payes attention to.
Contain the biologically active substances such as rich in protein, amino acid, organic acid, VITAMIN, unsaturated fatty acids and peptide antibiotics in the thalline of yeast saccharomyces cerevisiae and Lactobacterium acidophilum and secretory product thereof.Therefore, higher using value is all arranged in livestock and poultry cultivation and aquaculture.
At present, in livestock and poultry cultivation and aquaculture, in the face of the producers and consumers, two large problems being arranged is also two hang-ups: 1. protein utilization is not high; 2. the microbiotic use is excessive.Above-mentioned two problems and a difficult problem put forward and the present invention is exactly for solving.
Product function of the present invention is: 1. improve efficiency of feed utilization, allow the animals eat fermented product; 2. process the stomach of cultivated animals with biotechnological formulation, adjust colony balance, the kill harmful bacterium reduces antibiotic usage quantity; 3. use this product can significantly improve breeding environment.
Production technique of the present invention is simple, and application effect of products is good, can be widely used in the cultivation industries such as aquatic products, livestock and poultry.
Yeast saccharomyces cerevisiae and Lactobacterium acidophilum are used in the cultivation industries such as aquatic products, livestock and poultry, can improve fecundity and the survival ability of cultivated animals, can promote individual growth, strengthening immunity, disease resistance and integumentary musculature color and luster, can promote nutrition digestion to absorb.Can also the purifying aquaculture environment, purify water, the pollutents such as decomposing organic matter, hydrogen sulfide, amide.Nontoxic, harmless, without any side effects, benefit the nation and the people, be a kind of biological feed additive of green.Have wide market outlook.
At present, Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences from the ocean separation, purifying, identify many Accharomyces cerevisiaes and Lactobacterium acidophilum, through domestication, cultivate, improve and obtain that many strains product content is high, yeast saccharomyces cerevisiae and the Lactobacterium acidophilum bacterial classification of active good, fast growth, through our production application, its effect is very good, at Shenyang, the Dalian Area in Liaoning, the regional widespread use such as Hebei and Henan, obtained good economic benefit, we with Realization of Product commercialization production.
Summary of the invention
The object of the invention is to be to provide the feed of a kind of yeast saccharomyces cerevisiae and Lactobacterium acidophilum with the production method of complex microorganism preparations.
For achieving the above object, the technical solution used in the present invention is:
it is conventional S. cervisiae and Lactobacterium acidophilum that the present invention adopts bacterial classification, and with glucose, sucrose, sugar cane molasses, corn steep liquors etc. are main fermentation raw material, with the S. cervisiae of routine and Lactobacterium acidophilum bacterial classification under 30 ℃～34 ℃ aerobic conditions, separate, purifying, cultivate the production of hybrid seeds, cultivate through the solid first order seed, the liquid two stage fermentation culture, again with yeast saccharomyces cerevisiae mother bacterial liquid and Lactobacterium acidophilum mother liquor, mixed with the volume ratio in 1: 1 and produce the feed complex microorganism preparations product that contains yeast saccharomyces cerevisiae (cfu) 〉=2,500,000,000/ml and Lactobacterium acidophilum (cfu) 〉=5,000,000,000 viable bacteria/ml.The bacterium number of product is high, quality better, ph value 5.0～6.6,18 months quality guaranteed perioves.
The feed of a kind of yeast saccharomyces cerevisiae and Lactobacterium acidophilum comprises the steps: with the production method of complex microorganism preparations
1) single bacterium colony separates with preferred: with activated yeast saccharomyces cerevisiae slant strains, be coated with on the culture dish of the new microzyme culture medium that configures, through cultivating, obtain yeast saccharomyces cerevisiae list bacterium colony seed.With activated Lactobacterium acidophilum slant strains, be coated with on the culture dish of the new milk-acid bacteria substratum that configures, through cultivating, obtain the single bacterium colony seed of Lactobacterium acidophilum;
2) the yeast saccharomyces cerevisiae list bacterium colony seed and the yeast saccharomyces cerevisiae list bacterium colony seed that obtain respectively solid first order seed cultivation: with step 1), be inoculated into respectively in yeast and milk-acid bacteria one-level solid seed culture medium, solid seed culture bottle is the flat bottle of 200ml eggplant shape, through cultivating, obtain respectively yeast saccharomyces cerevisiae one-level solid seed and Lactobacterium acidophilum one-level solid seed;
3) the one-level solid seed that obtains respectively liquid two stage fermentation culture: with step 2) (the flat bottle of eggplant shape) is inoculated into respectively in second order fermentation tank liquid nutrient medium.One-level solid seed (the flat bottle of eggplant shape) with the inoculum size of second order fermentation tank liquid seeds nutrient solution is: one-level solid seed (the flat bottle of eggplant shape) is 1 bottle with the liquid weight ratio: 100 kilograms, through cultivating, obtain respectively secondary liquid fermenting mother liquor;
4) S. cervisiae (centrifugal through whizzer) mother liquid concentration (cfu) 〉=50/ml.
5) Lactobacterium acidophilum mother liquid concentration (cfu) 〉=10,000,000,000/ml.
The weight of the S. cervisiae seed culture medium that step 1) and 2) adopts consists of: glucose 2.0～3.0%, peptone 0.8.～1.2%, yeast extract paste 0.8.～1.2%, sodium-chlor 0.03～0.07%, sal epsom 0.03～0.07%, potassium primary phosphate 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, surplus are pure water, PH6.0～6.8, agar 1.5～2.2%;
Sterilising conditions: 0.08MPa, 105 ℃, 18min;
Culture condition: 26 ℃～32 ℃, time 48～72h.
The weight of the yeast saccharomyces cerevisiae bacteria fermentation culture medium that step 3) adopts consists of: sucrose 1.5～2.5%, sugar cane molasses 1.5～2.5%, peptone 0.2～0.8%, yeast powder 0.2～0.8%, corn steep liquor 2.0～4.0%, sodium-chlor 0.03～0.07%, sal epsom 0.03～0.07%, potassium primary phosphate 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, surplus are pure water, PH6.0～6.8;
Sterilising conditions: 0.08MPa, 105 ℃, 20min;
Fermentation culture conditions: tank pressure 0.01-0.10Mpa, 100: 10～120vvm of air quantity, rotating speed 220-260rpm, 26 ℃～32 ℃ of temperature, time 48～72h.
The weight of the Lactobacterium acidophilum seed culture medium that step 1) and 2) adopts consists of: glucose 0.5～1.5%, peptone 0.8～1.2%, extractum carnis 0.8～1.2%, yeast extract paste 0.3～0.7g, sodium-chlor 0.03～0.07%, sal epsom 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, calcium carbonate 0.5～1.5%, tomato juice 1～10%, surplus are pure water, PH5.6～6.8, agar 1.5～2.2%;
Sterilising conditions: 0.08MPa, 105 ℃, 18min;
Culture condition: 28 ℃～34 ℃ of temperature, time 48～72h.
The weight of the Lactobacterium acidophilum fermention medium that step 3) adopts consists of: glucose 0.5～1.5%, sugar cane molasses 0.5～1.5%, peptone 0.2～0.8%, yeast powder 0.2～0.8%, corn steep liquor 0.5～1.5%, sodium-chlor 0.03～0.07%, sal epsom 0.03～0.07%, potassium primary phosphate 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, calcium carbonate 0.5～1.5%, tomato juice 0～10%, surplus are pure water, PH5.6～6.8;
Sterilising conditions: 0.08MPa, 105 ℃, 18min;
Fermentation culture conditions is: tank pressure 0.01-0.10Mpa, 100: 0～100vvm of air quantity, rotating speed 0-100rpm, 30 ℃～38 ℃ of temperature, time are 48～72h.
Preferred version is as follows:
The weight of the S. cervisiae seed culture medium that adopts consists of: glucose 2.5%, peptone 1.0%, yeast extract paste 1.0%, sodium-chlor 0.05%, sal epsom 0.05%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.1%, surplus are pure water, PH6.8;
Sterilising conditions: 0.08MPa, 105 ℃, 18min;
Culture condition: 30 ℃ of temperature, time 64h..
The weight of the Lactobacterium acidophilum seed culture medium that adopts consists of: glucose 1.0%, peptone 1.0%, extractum carnis 1.0%, yeast extract paste 0.5g, sodium-chlor 0.05%, sal epsom 0.05%, dipotassium hydrogen phosphate 0.1%, calcium carbonate 1.0%, tomato juice 5%, surplus are pure water, PH6.8, agar 2.0%;
Sterilising conditions: 0.08MPa, 105 ℃, 18min;
Culture temperature is 34 ℃, and the time is 64h.
The weight of the yeast saccharomyces cerevisiae bacteria fermentation culture medium that adopts consists of: sucrose 2.0%, sugar cane molasses 2.0%, peptone 0.5%, yeast powder 0.5%, corn steep liquor 3.0%, sodium-chlor 0.05%, sal epsom 0.05%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.10%, surplus are pure water, PH6.8;
Sterilising conditions: 0.08MPa, 105 ℃, 20min;
Fermentation culture conditions is: tank pressure 0.05Mpa, air quantity 100: 100vvm, rotating speed 220rpm, 30 ℃ of temperature, time are 64h.
The weight of the Lactobacterium acidophilum fermention medium that adopts consists of: glucose 1.0%, sugar cane molasses 1.0%, peptone 0.5%, yeast powder 0.5%, corn steep liquor 1.0%, sodium-chlor 0.05%, sal epsom 0.05%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.10%, calcium carbonate 1.0%, tomato juice 5.0%, surplus are pure water, PH6.8.
Sterilising conditions: 0.08MPa, 105 ℃, 18min;
Fermentation culture conditions is: tank pressure 0.05Mpa, air quantity 100: 50vvm, rotating speed 50r pm, 34 ℃ of culture temperature, time are 64h.
Adopt colony counting method to check the feed complex microorganism preparations product of yeast saccharomyces cerevisiae and Lactobacterium acidophilum, contain yeast saccharomyces cerevisiae (cfu) 〉=2,500,000,000/ml and Lactobacterium acidophilum (cfu) 〉=5,000,000,000 viable bacteria/ml.
The feed of described a kind of yeast saccharomyces cerevisiae and Lactobacterium acidophilum can be widely used in the cultivation industries such as aquatic products, livestock and poultry with the product of complex microorganism preparations.
Production technique provided by the invention is simple, and the quality product of production is high, and application effect of products is good, and product is used in the cultivation such as aquatic products, livestock and poultry, can strengthening immunity, improve surviving rate, and enhancing development promotes quality, can improve and the purifying aquaculture environment.
The present invention has following advantage:
1. bacterial classification is good, and yeast saccharomyces cerevisiae and Lactobacterium acidophilum all have the characteristics of self, 1) nutritious, can secrete and the enzyme products such as the various proteolytic enzyme of metabolism, amylase and cellulase each seed amino acid, lactic acid, acetic acid, various organic acid and peptide antibiotics.2) these two culture propagation speed are fast, require not harsh to substratum and fermentation condition.3) all can be good at survival under acidic conditions, be easy to preservation.
2. the yeast saccharomyces cerevisiae of the present invention's employing and Lactobacterium acidophilum kind are the conventional bacterial classification of using, and take glucose, sugar cane molasses, corn steep liquor etc. as main fermentation raw material, at 30 ℃～34 ℃, carry out fermentation culture under aerobic condition, under the isometric growth condition, use cultural method of the present invention, can make yeast saccharomyces cerevisiae and the more general collective media of Lactobacterium acidophilum reproduction speed fast 1-2 days, bacteria concentration improves more than 30%.
3. culture medium raw material forms simple, wide material sources, cost is low.It is simply general that the yeast saccharomyces cerevisiae that the present invention adopts and Lactobacterium acidophilum are cultivated raw material, during production in enormous quantities, urban industry product tankage, agricultural byproducts secondary products be can also adopt, through comprehensive treating process, feed yeast saccharomyces cerevisiae and Lactobacterium acidophilum product can be made into after cultivation.
4. the feed of yeast saccharomyces cerevisiae and Lactobacterium acidophilum is with the good product quality of complex microorganism preparations.Take yeast saccharomyces cerevisiae and Lactobacterium acidophilum as provenance, the fermentation mother liquor of making, thalline quantity is many, and meta-bolites is many.Centrifugal through the cultivation of solid first order seed, liquid two stage seed fermentation, whizzer, 1; 1 mixes, and waits the production technique of the yeast saccharomyces cerevisiae produced and the product of the feed use complex microorganism preparations of Lactobacterium acidophilum, and easy and simple to handle, the cycle is short, good product quality, and effect is good
The yeast saccharomyces cerevisiae that application the present invention produces and the feed of Lactobacterium acidophilum the product of complex microorganism preparations, application test through in aquatic products, livestock and poultry cultivation shows the immunizing power that can strengthen animal body, can improve surviving rate, promote growth, improve quality; Can improve breeding environment, purify water, environmentally friendly.Deeply be subjected to the cultivation users' such as aquatic products, livestock and poultry welcome, be easy to apply.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
The feed of a kind of yeast saccharomyces cerevisiae and Lactobacterium acidophilum the production method of complex microorganism preparations can follow these steps to concrete operations:
Embodiment 1:
The inspection of the single bacterium colony Pureization of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) (producing proteolytic enzyme, amino acid, VITAMIN, vitamin H), the cultivation of solid first order seed, liquid two stage fermentation culture, bacterium number and definite, concrete operation step is as follows:
1) single bacterium colony separates with preferred: with activated yeast saccharomyces cerevisiae slant strains, be coated with on the culture dish of the new microzyme culture medium that configures, through cultivating, obtain yeast saccharomyces cerevisiae list bacterium colony seed.
Yeast saccharomyces cerevisiae list bacterium colony Pureization consists of with preferred culture medium: 20% potato (potato) leach liquor 1000mL, sucrose 20g, agar 18g, PH6.8; Also can use yeast saccharomyces cerevisiae one-level solid seed culture medium.
2) the yeast saccharomyces cerevisiae list bacterium colony seed that obtains solid first order seed cultivation: with upper step 1) is inoculated in yeast one-level solid seed culture medium, and solid seed culture bottle is the flat bottle of 200ml eggplant shape, through cultivating, obtains yeast saccharomyces cerevisiae one-level solid seed;
3) liquid two stage fermentation culture (selecting substratum and fermentating condition test): take tank fermentation method to produce fermented liquid, with the flat bottle of cultured eggplant shape yeast saccharomyces cerevisiae one-level solid seed, wash to the aseptic triangular flask of 500mL with the 200mL sterilized water, be inoculated in fermentor tank with flame inoculation method differential pressure, (fermentor tank can be 500 liters, 1 ton, 2 tons), liquid amount is no more than 70%, and aerobic fermentation is cultivated.Cultivate when ripe, bacterium liquid has a kind of wine flavour, and test under microscope S. cervisiae thalline is healthy and strong, without unusual phenomenoies such as living contaminantses, can for centrifugal and with mix mother liquor.
The S. cervisiae seed culture medium is selected test
Test design is 7 factor 3 levels, test design and test-results such as following table 1:
Table 1. S. cervisiae (triangular flask) culture medium prescription composite test
By the test-results of table 1, definite substratum is: glucose 25g, peptone 10g, yeast extract paste 10g, sodium-chlor 0.5g, sal epsom 0.5g, potassium primary phosphate 0.5g, dipotassium hydrogen phosphate 1g, 1000ml pure water, PH6.8; 0.08MPa, 105 ℃ the sterilization 18min; Culture condition: 30 ℃ of temperature, time 64h.
The S. cervisiae fermentation tank culture medium is selected test
The fermentation tank culture medium test design is 6 factor 3 levels, test design and test-results such as following table 2:
The test of table 2. S. cervisiae fermentation tank culture medium formula combination
By the test-results of table 2, definite S. cervisiae fermentation tank culture medium is: sucrose 20g, sugar cane molasses 20g, peptone 5g, yeast powder 5g, corn steep liquor 30g, sodium-chlor 0.5g, sal epsom 0.5g, potassium primary phosphate 0.5g, dipotassium hydrogen phosphate 1g, 1000ml pure water; 0.08MPa, 105 ℃ the sterilization 20 minutes.
The test of S. cervisiae fermentation culture conditions
The ferment tank condition test is designed to 5 factor 3 levels, test design and test-results such as following table 3:
The test of table 3. S. cervisiae tank fermentation culture conditions
By table 3 test-results, the fermentation condition of fermentor tank is defined as: tank pressure 0.05Mpa, air quantity 100: 100vvm, rotating speed 220rpm, 30 ℃ of temperature, time 64h.
4) S. cervisiae microscopy: it is most for unicellular microorganism that yeast cell form size is about 2-5 * 5-30 μ m yeast, often is oval or cylindrical.
5) the S. cervisiae culture dish checks: the granular projection that is white in color of the yeast colony on plate culture medium has wine flavour.
6) S. cervisiae shaking flask condition is: rotating speed is 30 ℃ of 150r/mil temperature.
Embodiment 2:
Lactobacterium acidophilum (Lactobacillus acidophilus), (producing lactic acid, acetic acid and various organic acid, lactobacillin, peptide antibiotics, proteolytic enzyme, VITAMIN etc.) single bacterium colony Pureization and preferred, the cultivation of solid first order seed, liquid two stage fermentation culture concrete operation step are as follows:
1) single bacterium colony Pureization is with preferred: with activated Lactobacterium acidophilum slant strains, be coated with on the culture dish of the new milk-acid bacteria substratum that configures, through cultivating, obtain the single bacterium colony seed of Lactobacterium acidophilum.
2) the single bacterium colony seed of the Lactobacterium acidophilum that obtains solid first order seed cultivation: with upper step 1), be inoculated in Lactobacterium acidophilum one-level solid seed culture medium, solid seed culture bottle is the flat bottle of 200ml eggplant shape, through cultivating, obtains Lactobacterium acidophilum one-level solid seed;
3) liquid two stage fermentation culture: its process is with the present embodiment 1 (2); Its difference culture temperature is 34 ℃, also can reach above-mentioned technical indicator, namely can use as mother liquor.
Fermentor tank inoculation and fermentation culture: take tank fermentation method to produce fermented liquid, with the flat bottle of cultured eggplant shape Lactobacterium acidophilum one-level solid seed, wash to the aseptic triangular flask of 500mL with the 200mL sterilized water, be inoculated in fermentor tank with flame inoculation method differential pressure, (fermentor tank can be 500 liters, 1 ton, 2 tons), liquid amount is no more than 70%, logical stingy amount fermentation culture, temperature is 34 ℃, and incubation time is 64 hours.The motion of test under microscope Lactobacterium acidophilum thalline is active, and stalwartness is neat, without miscellaneous bacteria, free from extraneous odour, the unusual phenomenon such as pollution-free, microscopy live lactobacillus acidophilus number can reach 10,000,000,000/ml (adopting counting method of blood cell to calculate the bacterium number), can stop fermentation, and fermented liquid namely can be used as mother liquor for mixing.
The Lactobacterium acidophilum seed culture medium is selected test
The Lactobacterium acidophilum seed culture medium is selected test, is designed to 7 factor 3 levels, test design and test-results such as following table 4:
Table 4. Lactobacterium acidophilum (triangular flask) culture medium prescription composite test
Table 4 test-results determines that substratum is: glucose 10g, peptone 10g, extractum carnis 10g, yeast extract paste 5g, sodium-chlor 0.5g, sal epsom 0.5g, dipotassium hydrogen phosphate 1.0g, calcium carbonate 10g, tomato juice 50g, 1000ml pure water, PH6.8; 0.08MPa, 105 ℃ the sterilization 18min; Culture condition is: 34 ℃ of temperature, time 64h.
The Lactobacterium acidophilum fermentation tank culture medium is selected test
Lactobacterium acidophilum fermentation tank culture medium test design is 7 factor 3 levels, test design and test-results such as following table 5:
The test of table 5. Lactobacterium acidophilum fermentation tank culture medium formula combination
The Lactobacterium acidophilum fermentor tank seed culture medium of determining by table 5 test-results is: glucose 10g, sugar cane molasses 10g, peptone 5g, yeast powder 5g, corn steep liquor 10g, sodium-chlor 0.5g, sal epsom 0.5g, dipotassium hydrogen phosphate 1.0g, calcium carbonate 10g, tomato juice 50g, 1000ml pure water, PH6.8; 0.08MPa, 105 ℃ the sterilization 20mil;
The test of Lactobacterium acidophilum tank fermentation culture conditions
Lactobacterium acidophilum tank fermentating condition test is designed to 5 factor 3 levels, test design and test-results such as following table 6:
Table 6. Lactobacterium acidophilum fermentor cultivation condition test
The Lactobacterium acidophilum fermentor cultivation condition of determining by table 6 test-results is: tank pressure 0.05Mpa, air quantity 100: 50vvm, rotating speed 50rpm, 34 ℃ of temperature, time are 64h.
Bacterium lacticum seed substratum consists of: glucose 10g, peptone 10g, extractum carnis 10g, yeast extract paste 5g, sodium acetate 5g, citric acid diamines 5g, sal epsom 0.2g, manganous sulfate 0.05g, dipotassium hydrogen phosphate 2g, calcium carbonate 20,1mL tween 80, water 1000mL, agar 20g, PH7.0
Embodiment 3:
The feed of yeast saccharomyces cerevisiae and Lactobacterium acidophilum is carried out application test with complex microorganism preparations in children's ginseng, the feed of yeast saccharomyces cerevisiae and Lactobacterium acidophilum sees Table 7 with complex microorganism preparations to the impact that sea cucumber increases weight;
The impact that the feed of table 7. yeast saccharomyces cerevisiae and Lactobacterium acidophilum increases weight on sea cucumber with the complex microorganism preparations sea cucumber
* test period: on March 15,1 day～2011 March in 2011
* water body: length * wide * height=1m * 1m * 1m=1m
3(cubic meter water body)
* test group 1 and test group each water body 2 every days are with 5 milliliters of complex microorganism preparations;
Described complex microorganism preparations is that following two kinds of mother liquors were mixed with the volume ratio in 1: 1; Two kinds of mother liquors are respectively:
Embodiment 1 step 3) the secondary liquid fermentation liquid of the S. cervisiae that obtains through the whizzer centrifugal concentrating, gets the yeast saccharomyces cerevisiae mother bacterial liquid, S. cervisiae mother liquid concentration (cfu) 〉=50/ml;
Embodiment 2 steps 3) obtain and the secondary liquid fermentation liquid of Lactobacterium acidophilum is the Lactobacterium acidophilum mother liquor Lactobacterium acidophilum mother liquid concentration (cfu) 〉=10,000,000,000/ml.
* control group each water body every day is with 5 milliliters of seawater;
* join 16 ℃ of pond water temperatures;
Can find out by table 7, when 16 ℃ of water temperatures, nursing through 15 days, test group 1 average growth rate 22.9, test group 2 average growth rates 21.5, control group average growth rate 17.7, test-results shows, the average growth rate of test group should add the complex microorganism preparations successful apparently higher than the control group average growth rate in holothruian cultures.
It is simple that the present invention has production technique, and easy handling, raw material sources are extensive, and cost is low to be easy to promote, tunning with short production cycle is many, and good product quality is easy to the characteristics such as application.Using in livestock and poultry and aquaculture, can improve immunizing power and surviving rate, can accelerate to grow and improve output, can purify water and improve breeding environment, is a kind of desirable high-quality microorganism feed addictive.
Embodiment 4
Be with embodiment 1-3 difference, for improving the quality of products, shorten the production cycle, it is better that the applicant has also tried out its effect of the high-activity microorganism bacterial strain that Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences's separation, purifying are also identified (as: yeast saccharomyces cerevisiae, Lactobacterium acidophilum and photosynthetic bacterium mixing are used).
Claims (10)
1. the feed of a yeast saccharomyces cerevisiae and Lactobacterium acidophilum with the production method of complex microorganism preparations, is characterized in that:
With the S. cervisiae fermentation culture, prepare the concentrated yeast saccharomyces cerevisiae mother bacterial liquid that contains yeast saccharomyces cerevisiae (cfu) 〉=5,000,000,000/ml through centrifugal;
The Lactobacterium acidophilum mother liquor that contains Lactobacterium acidophilum (cfu) 〉=10,000,000,000/ml of high density;
Again with yeast saccharomyces cerevisiae mother bacterial liquid and Lactobacterium acidophilum mother liquor, mixed with the volume ratio in 1: 1, produce the feed that contains yeast saccharomyces cerevisiae (cfu) 〉=2,500,000,000/ml, Lactobacterium acidophilum (cfu) 〉=5,000,000,000/ml with compound slight biotechnological formulation product.
2. according to production method claimed in claim 1, it is characterized in that:
Described yeast saccharomyces cerevisiae mother bacterial liquid is: with Wine brewing yeast strain, cultivate through the solid first order seed, the liquid two stage fermentation culture is through the yeast saccharomyces cerevisiae mother bacterial liquid of centrifugal concentrating acquisition;
Described Lactobacterium acidophilum mother liquor is: with lactobacillus acidophilus strains, cultivate liquid two stage fermentation culture, the Lactobacterium acidophilum mother liquor of acquisition through the solid first order seed;
Above-mentioned yeast saccharomyces cerevisiae mother bacterial liquid and Lactobacterium acidophilum mother liquor preparation process comprise the steps:
1) single bacterium colony separates with preferred: the S. cervisiae slant strains with activated, be coated with on the culture dish of microzyme culture medium, and through cultivating, obtain yeast saccharomyces cerevisiae list bacterium colony seed; Lactobacterium acidophilum slant strains with activated is coated with on the culture dish of milk-acid bacteria substratum, through cultivating, obtains the single bacterium colony seed of Lactobacterium acidophilum;
2) the single bacterium colony seed of the yeast saccharomyces cerevisiae list bacterium colony seed that obtains respectively solid first order seed cultivation: with step 1) and Lactobacterium acidophilum, be inoculated into respectively in yeast and milk-acid bacteria solid first order seed substratum, solid seed culture bottle is the flat bottle of 200ml eggplant shape, through cultivating, obtain respectively yeast saccharomyces cerevisiae one-level solid seed and Lactobacterium acidophilum one-level solid seed;
3) the one-level solid seed that obtains respectively liquid two stage fermentation culture: with step 2) (the flat bottle of eggplant shape) is inoculated into respectively in second order fermentation tank liquid nutrient medium; One-level solid seed (the flat bottle of eggplant shape) with the inoculum size of second order fermentation tank liquid seeds nutrient solution is: one-level solid seed (the flat bottle of eggplant shape) is 1 bottle with the liquid weight ratio: 100 kilograms, through cultivating, obtain respectively the secondary liquid fermentation liquid;
4) the secondary liquid fermentation liquid of S. cervisiae through the whizzer centrifugal concentrating, gets the yeast saccharomyces cerevisiae mother bacterial liquid, S. cervisiae mother liquid concentration (cfu) 〉=50/ml;
And the secondary liquid fermentation liquid of Lactobacterium acidophilum is the Lactobacterium acidophilum mother liquor, Lactobacterium acidophilum mother liquid concentration (cfu) 〉=10,000,000,000/ml.
3. according to production method claimed in claim 2, it is characterized in that:
The weight of the S. cervisiae seed culture medium that step 1) and 2) adopts consists of: glucose 2.0～3.0%, peptone 0.8.～1.2%, yeast extract paste 0.8.～1.2%, sodium-chlor 0.03～0.07%, sal epsom 0.03～0.07%, potassium primary phosphate 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, surplus are pure water, PH6.0～6.8, agar 1.5～2.2%; Sterilising conditions: 0.08MPa, 105 ℃, 18min;
Culture condition: 26 ℃～32 ℃, time 48～72h.
4. according to production method claimed in claim 3, it is characterized in that:
The weight of the S. cervisiae seed culture medium that adopts consists of: glucose 2.5%, peptone 1.0%, yeast extract paste 1.0%, sodium-chlor 0.05%, sal epsom 0.05%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.1%, surplus are pure water, PH6.8;
Sterilising conditions: 0.08MPa, 105 ℃, 18min;
Culture condition: 30 ℃ of temperature, time 64h..
5. according to production method claimed in claim 2, it is characterized in that:
The weight of the yeast saccharomyces cerevisiae bacteria fermentation culture medium that step 3) adopts consists of: sucrose 1.5～2.5%, sugar cane molasses 1.5～2.5%, peptone 0.2～0.8%, yeast powder 0.2～0.8%, corn steep liquor 2.0～4.0%, sodium-chlor 0.03～0.07%, sal epsom 0.03～0.07%, potassium primary phosphate 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, surplus are pure water, PH6.0～6.8;
Sterilising conditions: 0.08MPa, 105 ℃, 20min;
Fermentation culture conditions: tank pressure 0.01-0.10Mpa, 100: 10～120vvm of air quantity, rotating speed 220-260rpm, 26 ℃～32 ℃ of temperature, time 48～72h.
6. according to production method claimed in claim 5, it is characterized in that:
The weight of the yeast saccharomyces cerevisiae bacteria fermentation culture medium that adopts consists of: sucrose 2.0%, sugar cane molasses 2.0%, peptone 0.5%, yeast powder 0.5%, corn steep liquor 3.0%, sodium-chlor 0.05%, sal epsom 0.05%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.10%, surplus are pure water, PH6.8;
Sterilising conditions: 0.08MPa, 105 ℃, 20min;
Fermentation culture conditions is: tank pressure 0.05Mpa, air quantity 100: 100vvm, rotating speed 220rpm, 30 ℃ of temperature, time are 64h.
7. according to production method claimed in claim 2, it is characterized in that:
The weight of the Lactobacterium acidophilum seed culture medium that step 1) and 2) adopts consists of: glucose 0.5～1.5%, peptone 0.8～1.2%, extractum carnis 0.8～1.2%, yeast extract paste 0.3～0.7g, sodium-chlor 0.03～0.07%, sal epsom 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, calcium carbonate 0.5～1.5%, tomato juice 1～10%, surplus are pure water, PH5.6～6.8, agar 1.5～2.2%;
Sterilising conditions: 0.08MPa, 105 ℃, 18min;
Culture condition: 28 ℃～34 ℃ of temperature, time 48～72h.
8. according to production method claimed in claim 7, it is characterized in that:
The weight of the Lactobacterium acidophilum seed culture medium that adopts consists of: glucose 1.0%, peptone 1.0%, extractum carnis 1.0%, yeast extract paste 0.5g, sodium-chlor 0.05%, sal epsom 0.05%, dipotassium hydrogen phosphate 0.1%, calcium carbonate 1.0%, tomato juice 5%, surplus are pure water, PH6.8, agar 2.0%;
Sterilising conditions: 0.08MPa, 105 ℃, 18min;
Culture temperature is 34 ℃, and the time is 64h.
9. according to production method claimed in claim 2, it is characterized in that:
The weight of the Lactobacterium acidophilum fermention medium that step 3) adopts consists of: glucose 0.5～1.5%, sugar cane molasses 0.5～1.5%, peptone 0.2～0.8%, yeast powder 0.2～0.8%, corn steep liquor 0.5～1.5%, sodium-chlor 0.03～0.07%, sal epsom 0.03～0.07%, potassium primary phosphate 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, calcium carbonate 0.5～1.5%, tomato juice 0～10%, surplus are pure water, PH5.6～6.8;
Sterilising conditions: 0.08MPa, 105 ℃, 18min;
Fermentation culture conditions is: tank pressure 0.01-0.10Mpa, 100: 0～100vvm of air quantity, rotating speed 0-100rpm, 30 ℃～38 ℃ of temperature, time are 48～72h.
10. according to production method claimed in claim 9, it is characterized in that:
The weight of the Lactobacterium acidophilum fermention medium that adopts consists of: glucose 1.0%, sugar cane molasses 1.0%, peptone 0.5%, yeast powder 0.5%, corn steep liquor 1.0%, sodium-chlor 0.05%, sal epsom 0.05%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.10%, calcium carbonate 1.0%, tomato juice 5.0%, surplus are pure water, PH6.8;
Sterilising conditions: 0.08MPa, 105 ℃, 18min;
Fermentation culture conditions is: tank pressure 0.05Mpa, air quantity 100: 50vvm, rotating speed 50rpm, 34 ℃ of culture temperature, time are 64h.
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| CN103404716B (en) * | 2013-07-09 | 2015-08-19 | 青岛中浩环能技术开发有限公司 | Compound microorganism bacterium powder and preparation method |
| CN105861615A (en) * | 2016-04-22 | 2016-08-17 | 闫大凤 | Production method of amino acid bioactive peptide for aquatic products |
| CN107937306A (en) * | 2017-11-27 | 2018-04-20 | 上海创博生态工程有限公司 | A kind of microbe leaven for fermented liquid feed and preparation method thereof |
| CN108517345A (en) * | 2018-04-20 | 2018-09-11 | 杭州优玛达生物科技有限公司 | A kind of method of thermus thermophilus and saccharomycete combined fermentation |
| CN108991222A (en) * | 2018-06-29 | 2018-12-14 | 哈尔滨博善联合生物饲料有限公司 | The method of brick is licked using arginine fermentation secondary species production ruminant nutrition type |
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| CN109329648A (en) * | 2018-09-10 | 2019-02-15 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of application of the compound micro-ecological preparation of antagonism prawn vibrios |
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