CN103173371B - 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.For making bread and wine brewing, in modern molecule and cytobiology, be used as eucaryon model animals traditionally.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 intestinal microflora balance, suppresses the propagation of enteron aisle undesirable microorganism, and pathogenic microorganism is had to antagonistic action.Particularly to gastrointestinal dysfunction with take antibiotic and cause that the symptoms such as gastrointestinal dysfunction have special efficacy, can make rapidly the flora in enteron aisle recover normal equilibrium, suppress the propagation of spoilage organism, there is good health protection effect.
In research in the past, all there is comparatively deep research the aspects such as taxonomy, physiological and biochemical property, fermentation condition and the regulation and control thereof of scientific worker to yeast saccharomyces cerevisiae and Lactobacterium acidophilum.The applied research of yeast saccharomyces cerevisiae and Lactobacterium acidophilum is also obtained to very large progress.But to the interactive research between the two of yeast saccharomyces cerevisiae and Lactobacterium acidophilum and application report and few.Yeast saccharomyces cerevisiae and Lactobacterium acidophilum, both have common feature 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, being more and more subject to scientific worker and business people payes attention to.
In the thalline of yeast saccharomyces cerevisiae and Lactobacterium acidophilum and secretory product thereof, contain the biologically active substances such as rich in protein, amino acid, organic acid, VITAMIN, unsaturated fatty acids and peptide antibiotics.Therefore, in livestock and poultry cultivation and aquaculture, all there is higher using value.
At present, in livestock and poultry cultivation and aquaculture, having two large problems in the face of producers and consumers is also two hang-ups: 1. protein utilization is not high; 2. microbiotic use is excessive.And the present invention is exactly for solving, above-mentioned two problems and a difficult problem put forward.
Product function of the present invention is: 1. improve efficiency of feed utilization, allow animals eat fermented product; 2. process the stomach of cultivated animals with biotechnological formulation, adjust colony balance, 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 such as aquatic products, livestock and poultry industry.
Yeast saccharomyces cerevisiae and Lactobacterium acidophilum are applied in the cultivation such as aquatic products, livestock and poultry industry, 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 purifying aquaculture environment, purify water, the pollutent 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.There are wide market outlook.
At present, Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences from ocean separation, purifying, identify many Accharomyces cerevisiaes and Lactobacterium acidophilum, through domestication, cultivate, improve the yeast saccharomyces cerevisiae and the Lactobacterium acidophilum bacterial classification that obtain high, active good, the fast growth of many strains product content, through our production application, its effect is very good, in Shenyang, the Dalian Area in Liaoning, the regional widespread use such as Hebei and Henan, obtained good economic benefit, we by Realization of Product commercialization production.
Summary of the invention
The object of the invention is the production method of the feed complex microorganism preparations that is to provide a kind of yeast saccharomyces cerevisiae and Lactobacterium acidophilum.
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, by conventional S. cervisiae and Lactobacterium acidophilum bacterial classification under 30 ℃～34 ℃ aerobic conditions, separate, purifying, cultivate the production of hybrid seeds, cultivate through solid first order seed, liquid two stage fermentation culture, again by yeast saccharomyces cerevisiae mother bacterial liquid and Lactobacterium acidophilum mother liquor, mix 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 in 1: 1 same volume ratio.The bacterium number of product is high, quality better, ph value 5.0～6.6,18 months quality guaranteed perioves.
A production method for complex microorganism preparations for the feed of yeast saccharomyces cerevisiae and Lactobacterium acidophilum, comprises the steps:
1) single bacterium colony separates with preferred: by activated yeast saccharomyces cerevisiae slant strains, on the culture dish of the new microzyme culture medium configuring, be coated with, through cultivating, obtain yeast saccharomyces cerevisiae list bacterium colony seed.By activated Lactobacterium acidophilum slant strains, on the culture dish of the new milk-acid bacteria substratum configuring, be coated with, through cultivating, obtain the single bacterium colony seed of Lactobacterium acidophilum;
2) solid first order seed is cultivated: by step 1) in the yeast saccharomyces cerevisiae list bacterium colony seed and the yeast saccharomyces cerevisiae list bacterium colony seed that obtain respectively, 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) liquid two stage fermentation culture: by step 2) in the one-level solid seed (eggplant shape flat bottle) that obtains be respectively 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 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.
Step 1) and 2) weight of the S. cervisiae seed culture medium that 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%, magnesium sulfate 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.
Step 3) weight of the yeast saccharomyces cerevisiae bacteria fermentation culture medium that 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%, magnesium sulfate 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.
Step 1) and 2) weight of the Lactobacterium acidophilum seed culture medium that 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%, magnesium sulfate 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.
Step 3) weight of the Lactobacterium acidophilum fermention medium that 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%, magnesium sulfate 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 adopting consists of: glucose 2.5%, peptone 1.0%, yeast extract paste 1.0%, sodium-chlor 0.05%, magnesium sulfate 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 adopting consists of: glucose 1.0%, peptone 1.0%, extractum carnis 1.0%, yeast extract paste 0.5g, sodium-chlor 0.05%, magnesium sulfate 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 adopting 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%, magnesium sulfate 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 adopting 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%, magnesium sulfate 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 product of complex microorganism preparations for described a kind of yeast saccharomyces cerevisiae and the feed of Lactobacterium acidophilum, can be widely used in the cultivation such as aquatic products, livestock and poultry industry.
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 application in the cultivation such as aquatic products, livestock and poultry, can strengthening immunity, improves surviving rate, and enhancing development, promotes quality, can improve and purifying aquaculture environment.
Tool of the present invention has the following advantages:
1. bacterial classification is good, and yeast saccharomyces cerevisiae and Lactobacterium acidophilum all have the feature of self, 1) nutritious, can secrete and the enzyme product 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 under acidic conditions, can be good at survival, be easy to preservation.
2. the present invention adopts yeast saccharomyces cerevisiae and Lactobacterium acidophilum kind are routine bacterial classification, and take glucose, sugar cane molasses, corn steep liquor etc. as main fermentation raw material, at 30 ℃～34 ℃, under aerobic condition, carry out fermentation culture, under isometric growth condition, apply cultural method of the present invention, can make yeast saccharomyces cerevisiae and the fast 1-2 days of the more general collective media of Lactobacterium acidophilum reproduction speed, bacteria concentration improves more than 30%.
3. simple, the wide material sources of culture medium raw material composition, cost are low.It is simply general that the yeast saccharomyces cerevisiae that the present invention adopts and Lactobacterium acidophilum are cultivated raw material, when 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 after cultivation, can be made into.
4. the good product quality of complex microorganism preparations for the feed of yeast saccharomyces cerevisiae and Lactobacterium acidophilum.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 product of the feed complex microorganism preparations of the yeast saccharomyces cerevisiae that application the present invention produces and Lactobacterium acidophilum, through the application test in aquatic products, livestock and poultry cultivation, show 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 subject to the cultivation such as aquatic products, livestock and poultry user's welcome, be easy to apply.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
A production method for complex microorganism preparations for the feed of yeast saccharomyces cerevisiae and Lactobacterium acidophilum, 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: by activated yeast saccharomyces cerevisiae slant strains, on the culture dish of the new microzyme culture medium configuring, be coated with, 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) solid first order seed is cultivated: by upper step 1) in the yeast saccharomyces cerevisiae list bacterium colony seed that obtains, be inoculated in yeast one-level solid seed culture medium, 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, by flat cultured eggplant shape bottle yeast saccharomyces cerevisiae one-level solid seed, wash to the aseptic triangular flask of 500mL with 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, test under microscope S. cervisiae thalline stalwartness, without unusual phenomenoies such as living contaminantses, can for centrifugal and with mix mother liquor.
S. cervisiae seed culture medium is selected test
Test design is 7 factor 3 levels, and test design and test-results are 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, magnesium sulfate 0.5g, potassium primary phosphate 0.5g, dipotassium hydrogen phosphate 1g, 1000ml pure water, PH6.8; 0.08MPa, 105 ℃ of sterilizing 18min; Culture condition: 30 ℃ of temperature, time 64h.
S. cervisiae fermentation tank culture medium is selected test
Fermentation tank culture medium test design is 6 factor 3 levels, and test design and test-results are 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, magnesium sulfate 0.5g, potassium primary phosphate 0.5g, dipotassium hydrogen phosphate 1g, 1000ml pure water; 0.08MPa, 105 ℃ of sterilizings 20 minutes.
The test of S. cervisiae fermentation culture conditions
Ferment tank condition test is designed to 5 factor 3 levels, and test design and test-results are 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, is often oval or cylindrical.
5) 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 with preferably, the cultivation of solid first order seed, liquid two stage fermentation culture concrete operation step be as follows:
1) single bacterium colony Pureization is with preferred: by activated Lactobacterium acidophilum slant strains, on the culture dish of the new milk-acid bacteria substratum configuring, be coated with, through cultivating, obtain the single bacterium colony seed of Lactobacterium acidophilum.
2) solid first order seed is cultivated: by upper step 1) in the single bacterium colony seed of the Lactobacterium acidophilum that obtains, 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, can be used as mother liquor and uses.
Fermentor tank inoculation and fermentation culture: take tank fermentation method to produce fermented liquid, by flat cultured eggplant shape bottle Lactobacterium acidophilum one-level solid seed, wash to the aseptic triangular flask of 500mL with 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, healthy and strong, neatly, 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 bacterium number), can stop fermentation, fermented liquid can be used as mother liquor for mixing.
Lactobacterium acidophilum seed culture medium is selected test
Lactobacterium acidophilum seed culture medium is selected test, is designed to 7 factor 3 levels, and test design and test-results are 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, magnesium sulfate 0.5g, dipotassium hydrogen phosphate 1.0g, calcium carbonate 10g, tomato juice 50g, 1000ml pure water, PH6.8; 0.08MPa, 105 ℃ of sterilizing 18min; Culture condition is: 34 ℃ of temperature, time 64h.
Lactobacterium acidophilum fermentation tank culture medium is selected test
Lactobacterium acidophilum fermentation tank culture medium test design is 7 factor 3 levels, and test design and test-results are 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 definite 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, magnesium sulfate 0.5g, dipotassium hydrogen phosphate 1.0g, calcium carbonate 10g, tomato juice 50g, 1000ml pure water, PH6.8; 0.08MPa, 105 ℃ of sterilizing 20mil;
The test of Lactobacterium acidophilum tank fermentation culture conditions
Lactobacterium acidophilum tank fermentating condition test is designed to 5 factor 3 levels, and test design and test-results are as following table 6:
Table 6. Lactobacterium acidophilum fermentor cultivation condition test
The Lactobacterium acidophilum fermentor cultivation condition definite 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, magnesium sulfate 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 to application test with complex microorganism preparations in children's ginseng, and the impact that the feed of yeast saccharomyces cerevisiae and Lactobacterium acidophilum increases weight on sea cucumber with complex microorganism preparations is in table 7;
The feed of table 7. yeast saccharomyces cerevisiae and Lactobacterium acidophilum is the impact on sea cucumber weightening finish with 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 2 every day of test group 5 milliliters of complex microorganism preparations for each water body;
Described complex microorganism preparations is that following two kinds of mother liquors are mixed in 1: 1 same volume ratio; Two kinds of mother liquors are respectively:
Embodiment 1 step 3) the secondary liquid fermentation liquid of the S. cervisiae that obtains is through whizzer centrifugal concentrating, obtains 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 Lactobacterium acidophilum mother liquor, Lactobacterium acidophilum mother liquid concentration (cfu) >=10,000,000,000/ml.
* control group 5 milliliters of seawater for each water body every day;
* join 16 ℃ of pond water temperatures;
Can find out by table 7, in the time of 16 ℃ of water temperatures, through the nursing of 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, apparently higher than control group average growth rate, should add complex microorganism preparations successful 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 features such as application.In livestock and poultry and aquaculture, applying, 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 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. a production method for complex microorganism preparations for the feed of yeast saccharomyces cerevisiae and Lactobacterium acidophilum, is characterized in that:
By S. cervisiae fermentation culture, prepare the concentrated yeast saccharomyces cerevisiae mother bacterial liquid containing yeast saccharomyces cerevisiae cfu >=5,000,000,000/ml through centrifugal;
The Lactobacterium acidophilum mother liquor containing Lactobacterium acidophilum cfu >=10,000,000,000/ml of high density;
Again by yeast saccharomyces cerevisiae mother bacterial liquid and Lactobacterium acidophilum mother liquor, mix in 1:1 same volume ratio, produce the feed complex microorganism preparations product that contains yeast saccharomyces cerevisiae cfu >=2,500,000,000/ml, Lactobacterium acidophilum cfu >=5,000,000,000/ml;
Described feed is applicable to the body weight gain of sea cucumber meat ginseng with complex microorganism preparations product.
2. according to production method claimed in claim 1, it is characterized in that:
Described yeast saccharomyces cerevisiae mother bacterial liquid is: by Wine brewing yeast strain, cultivate liquid two stage fermentation culture, the yeast saccharomyces cerevisiae mother bacterial liquid obtaining through centrifugal concentrating through solid first order seed;
Described Lactobacterium acidophilum mother liquor is: by lactobacillus acidophilus strains, cultivate liquid two stage fermentation culture, the Lactobacterium acidophilum mother liquor of acquisition through 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: by activated S. cervisiae slant strains, on the culture dish of microzyme culture medium, be coated with, through cultivating, obtain yeast saccharomyces cerevisiae list bacterium colony seed; By activated Lactobacterium acidophilum slant strains, on the culture dish of milk-acid bacteria substratum, be coated with, through cultivating, obtain the single bacterium colony seed of Lactobacterium acidophilum;
2) solid first order seed is cultivated: by the yeast saccharomyces cerevisiae list bacterium colony seed obtaining respectively in step 1) and the single bacterium colony seed of 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) liquid two stage fermentation culture: by step 2) in the one-level solid seed that obtains be respectively inoculated into respectively in second order fermentation tank liquid nutrient medium; The inoculum size of one-level solid seed and second order fermentation tank liquid seeds nutrient solution is: one-level solid seed is 1 bottle with liquid weight ratio: 100 kilograms, through cultivating, obtain respectively secondary liquid fermentation liquid;
4) the secondary liquid fermentation liquid of S. cervisiae, through whizzer centrifugal concentrating, obtains yeast saccharomyces cerevisiae mother bacterial liquid, S. cervisiae mother liquid concentration cfu >=50/ml;
And the secondary liquid fermentation liquid of Lactobacterium acidophilum is 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:
Step 1) and 2) weight of the S. cervisiae seed culture medium that 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%, magnesium sulfate 0.03～0.07%, potassium primary phosphate 0.03～0.07%, dipotassium hydrogen phosphate 0.05～0.15%, agar 1.5～2.2%, surplus are pure water; PH6.0～6.8, sterilising conditions: 0.08MPa, 105 ℃, 18 min;
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 adopting consists of: glucose 2.5%, peptone 1.0%, yeast extract paste 1.0%, sodium-chlor 0.05%, magnesium sulfate 0.05%, potassium primary phosphate 0.05%, dipotassium hydrogen phosphate 0.1%, surplus are pure water, PH6.8;
Sterilising conditions: 0.08MPa, 105 ℃, 18 min;
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%, magnesium sulfate 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, air quantity 100:10～120vvm, 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 adopting 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%, magnesium sulfate 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:
Step 1) and 2) weight of the Lactobacterium acidophilum seed culture medium that 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%, magnesium sulfate 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 ℃, 18 min;
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 adopting consists of: glucose 1.0%, peptone 1.0%, extractum carnis 1.0%, yeast extract paste 0.5g, sodium-chlor 0.05%, magnesium sulfate 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 ℃, 18 min;
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%, magnesium sulfate 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 ℃, 18 min;
Fermentation culture conditions is: tank pressure 0.01-0.10Mpa, air quantity 100:0～100vvm, turn
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 adopting 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%, magnesium sulfate 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 ℃, 18 min;
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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| 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 |
| CN108517345B (en) * | 2018-04-20 | 2021-07-27 | 杭州优玛达生物科技有限公司 | Thermophilic thermus strain and microzyme combined fermentation method |
| 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 |
| CN109090337A (en) * | 2018-07-05 | 2018-12-28 | 江瀚生物科技（上海）有限公司 | A kind of additive for microbe feedstuff and preparation method thereof improving cultivated animals immunity of organisms |
| CN109329648B (en) * | 2018-09-10 | 2022-03-08 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of compound microecological preparation for antagonizing vibrio prawn |
| CN110637942A (en) * | 2019-10-31 | 2020-01-03 | 辽宁威兰生物技术有限责任公司 | Laying hen premix rich in amino acid eggs and production method and application thereof |
| CN111349581A (en) * | 2020-02-20 | 2020-06-30 | 福建省新闽科生物科技开发有限公司 | Breeding and application of acidophilic lactobacillus |
| CN111513202A (en) * | 2020-05-29 | 2020-08-11 | 中农极润生物科技(北京)有限公司 | Microecological preparation and preparation method and application thereof |
| CN112725202A (en) * | 2021-01-20 | 2021-04-30 | 北京英惠尔生物技术有限公司 | Saccharomyces cerevisiae and lactobacillus acidophilus co-culture, preparation method and application thereof, and fermented soybean meal |
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