CN101085982A - Preparation method for producing microbe polysaccharides formulation using lactobacillus and microzyme - Google Patents
Preparation method for producing microbe polysaccharides formulation using lactobacillus and microzyme Download PDFInfo
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- CN101085982A CN101085982A CN 200610027552 CN200610027552A CN101085982A CN 101085982 A CN101085982 A CN 101085982A CN 200610027552 CN200610027552 CN 200610027552 CN 200610027552 A CN200610027552 A CN 200610027552A CN 101085982 A CN101085982 A CN 101085982A
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Abstract
The invention relates to a method for preparing microbiological polysaccharide product by using lactic acid bacillus and yeast. It is characterized in that it employs liquid culture of benefitial bacteria, collects bacteria cell wall through centrifugation, solid enlargement culturing bacteria group, adding polysaccharide- rich cell wall into culture medium. The high- concentration lactic acid bacteria in product can distinctively improve digestion and absorption for aquatic animal, generated peptidoglycan increases immunity of aquatic animal to pathogenesis; high- concentration yeast in the product can increase single-cell protein amount needed for fish growth, the yeast cell is broke, and releases a large amount of beta- 1, 3- dextran, which can apparently increase immunocompetence for Gram-positive bacteria and Gram-negative bacteria. The invention employs grain crop and commonly used feedstuff carrier as main raw material for culture medium, which reduces production cost, and fermentation device and production equipment are commonly used, which is easy for spread in aquaculture.
Description
Technical field
The present invention relates to a kind of preparation method who utilizes lactobacillus and yeast to produce microbe polysaccharides formulation, can be used for improving the immunological competence of cultivated animals such as beasts, birds and aquatic products, belong to the agro-ecology product technique field.
Background technology
Flourish along with China aquaculture intensification, cultivation water environment pollutes serious day by day, meanwhile, the discharging of undressed breeding wastewater and industry, sanitary sewage makes water body be subjected to severe contamination, the breeding ecological environment is destroyed, cause the pathogenic micro-organism kind to increase with velocity of propagation and accelerate, the aquaculture organism disease is on the rise, and causes heavy losses for the aquatic products aquaculture.According to incompletely statistics, moderate above breed disease area takes place and accounts for 15%～20% of the breed total area in the annual whole nation, and production loss is above 1,000,000 tons.Because aquatic animal disease that environmental degradation causes or the communicable disease that causes thus increase severely in a large number, become the major obstacle that the restriction culture fishery develops in a healthy way.Life-time service microbiotic and the control of other chemicalses are cultured disease and have been caused a series of environment and social concern, and its outstanding behaviours is microbiotic and the residual increase of harmful chemical in the product, and the fishery products price remains inactive over long periods of time.Utilize the digest and assimilate ability of beneficial microorganism at water body and meta-bolites raising cultivated animals, suppress a large amount of breedings of pathogenic bacterium simultaneously, this is a kind of environmental treatment method of effecting a permanent cure, and also is the best measure of carrying out green cultivation.
Summary of the invention
The purpose of this invention is to provide a kind of preparation method who utilizes lactobacillus and yeast to produce microbe polysaccharides formulation who improves the cultivated animals immunological competence and digest and assimilate ability.
For realizing above purpose, technical scheme of the present invention provides a kind of preparation method who utilizes lactobacillus and yeast to produce microbe polysaccharides formulation, it is characterized in that, adopts liquid at high speed to cultivate and solid expansion bonded fermentation process, and its production stage is:
The first step: fermentation strain seed selection
A. bacterial strain screening: with secretory product in the crucian enteron aisle of health is the bacterium source, adopts MRS milk-acid bacteria substratum to carry out milk-acid bacteria and separates, and filters out a kind of plant lactobacillus, selects substratum to separate S. cervisiae with yeast in beer fermentation liquid; Isolating two kinds of bacterial classifications are placed on respectively in the yeast extract paste wort agar substratum, under 4 ℃ of temperature, preserve bacterial classification;
B. be inoculated in isolating plant lactobacillus and S. cervisiae in the broth culture respectively, under 35-40 ℃ of temperature, cultivated 20-25 hour, then S. cervisiae is inoculated in yeast extract paste wort agar substratum, plant lactobacillus is inoculated in the milk-acid bacteria substratum, carry out streak culturely, select that growth is fast, thalline evident characteristic plant lactobacillus, S. cervisiae preserve respectively in the laboratory;
C. the production production of hybrid seeds: will select the fast plant lactobacillus of growth at last and transfer in the eggplant bottle that nutrient agar is housed, the S. cervisiae yeast extract paste wort agar of transferring is done in the eggplant bottle of substratum, under 35-40 ℃ of temperature, cultivated 45-50 hour, treat that eggplant bottle surface lawn is covered with, and can take out when band is yellow in white, put into 2-6 ℃ refrigerator then and preserve;
Second step: the liquid culture of useful bacterial strain
A. take by weighing each bacterial classification by following weight proportion example: plant lactobacillus 55-65%, S. cervisiae 35-45%
B. the plant lactobacillus that takes by weighing and S. cervisiae are mixed and carry out compound cultivation: the mixing lawn that takes by weighing among the step a is in 1: the ratio of 25-30 is inoculated in 120-130 ℃, the liquid nutrient medium of 30-45min high-temperature sterilization, in fermentor tank, stir 30min, its rotating speed is 200-240r/min, put into sterilization back plastic tank after stirring and left standstill 5-7 days, leave standstill the gas of measuring Ph value and release output in the process every day;
C. sampling 3 times in the fermenting process, microscopically detects the thalli growth situation and measures Ph value, stops to ferment when 2,000,000,000/ml concentration and Ph value reach 3.0-4.0 when total viable count reaches;
The 3rd step: centrifugal collection somatic cells wall
A. with fermented liquid in the step 1 at whizzer with every 1kg8000 r/min, the centrifugal collection bacterial sediment of 10min, is suspended in thalline in the distilled water to white with 0.9% physiological saline repetitive scrubbing bacterium, handle 30-40min with the ultrasonic cell-break machine then, carry out physics broken 2-4 time;
B. cell walls separates: with the solution after the physics fragmentation at whizzer with the centrifugal 15-20min of every 1kg4000r/min, remove throw out, to remove not broken thalline, again with supernatant at whizzer with the centrifugal 20min of every 1kg8000r/min, the thickness cell wall of collecting precipitation;
The 4th step: the solid enlarged culturing of flora
A. the bacterium liquid with the compound cultivation of step 2 mixes in 1: in 120-130 ℃ of the ratio access of 25-30, the solid enlarged culturing base of 30-45min high-temperature sterilization, stir 30-45min to even, pour the 50-70kg sealing and fermenting in the double-deck feedbag of sterilizing into, temperature 30-35 ℃, time 8-10 days, sampling was three times in the fermenting process, measures moisture and Ph value, take a sample after the fermentation ends, mensuration moisture is that 35-40%, Ph value are 3.5-4.0;
B. add the thickness cell wall: cell walls gleanings and solid are enlarged fermented product by 1: the 50-60 mixed, be poured in the stirrer and stir, rotating speed is 150-200 commentaries on classics/min, churning time is 30min;
The 5th step: pulverize and add carrier
A. dry: the product of step 4 fermentation finished thoroughly1 is in chronological sequence put into the incubator drying, and temperature is controlled at 40-45 ℃, and time 14-16 hour, the material moisture content that drying is finished is controlled at≤and 12%;
B. add carrier: with dried product in 1: the ratio of 0.02-0.03 is added carrier, pour in the stirrer and stir, change pulverizer then over to and pulverize, temperature of charge is controlled at below 45 ℃, material after the pulverizing is crossed 50 eye mesh screens, and sieve back coarse fodder is pulverized again;
C. packing into after sieving has in the bucket or bag of inner bag, tightens sack, fastens Sign Board, changes stockyard over to and stacks in order; Sampling detects, significant parameter: moisture content≤12%, viable count 60-80 hundred million/g; Physical behavior: deep yellow pulverulent solids.
Described step 2 liquid nutrient medium is at least three kinds of mixtures that sterilized water adds bean cake powder, urea, ammonium sulfate, yeast powder, Semen Maydis powder, molasses, potassium primary phosphate, Sodium phosphate dibasic, trimagnesium phosphate, sal epsom again, and its weight percent proportioning is: dregs of beans 2-6%, urea 1-3%, ammonium sulfate 0-1%, yeast powder 0-3%, Semen Maydis powder 2-8%, molasses 10-20%, potassium primary phosphate 0-1%, SODIUM PHOSPHATE, MONOBASIC 0-2%, trimagnesium phosphate 0-1%, sal epsom 0-1%, sterilized water 54-84%.
The solid enlarged culturing base of described step 4 is that at least four kinds of mixtures that clear chaff and sterilized water add in bean cake powder, molasses, Semen Maydis powder, potassium primary phosphate, SODIUM PHOSPHATE, MONOBASIC, sal epsom, sterilized water 10-30%, the urea are again formed, and its weight percent proportioning is: dregs of beans 2-6%, molasses 8-12%, Semen Maydis powder 4-8%, clear chaff 40-70%, potassium primary phosphate 0.03-0.06%, SODIUM PHOSPHATE, MONOBASIC 1-2%, sal epsom 0.4-0.8%, urea 1-2%, sterilized water 15-45%.
Described carrier is made up of zeolite powder, lime carbonate, and its weight percent proportioning is: zeolite powder 40-50%, lime carbonate 50-60%.
Nonspecific defense mechanism is that the first line of defence of vertebrates opposing pathogenic agent infected by microbes means cells a large amount of in body tissue and the body fluid and antimicrobial protein, peptide class.In microorganism cells, exist peptide class and polysaccharide that these can directly be absorbed by cultivated animals equally, as peptidoglycan and dextran etc., peptidoglycan (PG) is present in the cell walls of gram-positive microorganism and Gram-negative bacteria, and being the disaccharides that formed by N-acetylglucosamine and-acetylmuramic acid intersects the polymkeric substance that constitutes mutually by tyre chain.Be proved oral peptidoglycan and can have strengthened the resistivity of fish the Gram-positive germ, peptidoglycan can also strengthen the leukocytic phagocytosis of prawn and to the resistivity (Itami er al.1998) of white spot syndrome virus, and antibiotic, the bacteriolyze in the prawn hemolymph, phenol oxidase and superoxide dismutase are had in various degree inducing action.(Meng Fan is superfine, 1999).The immune effect of dextran has obtained extensive studies, now be applied to the yeast glucan that has in the aquatic animal, β-1, types such as 3 dextran, wherein studying maximum is yeast glucan, yeast glucan can improve N,O-Diacetylmuramidase output of fish and the fungicidal activity of scavenger cell (Jorgensen, Rorstad, 1993).β-1,3 dextran has been used to preventing disease, and it can strengthen N,O-Diacetylmuramidase and the complement activity of fish, the output of cytophagous breathing explosive action and superoxide thereof.Generally speaking, microbial polysaccharide is the biological polymer that a class has multiple complicated group, it can activate the non-specific immunity system as immunostimulant, and these polymkeric substance are the particular sequence structures of evolving and remaining by microorganism usually, but are not the moietys of higher animal body.
This patent is chosen a kind of lactobacillus from animal intestinal, carry out shake flat experiment, be chosen in the bacterial classification that the substratum high speed is produced, simultaneously in the beer fermentation substrate, choose a kind of S. cervisiae, contain particularly β-1,3 dextran of a large amount of dextran in its cell walls, can significantly improve the immunological competence of cultivated animals to Gram-positive and negative germ, improve the digest and assimilate ability of animal to feed simultaneously, day weight gain is obviously improved, the culture-cycle shortens.
Advantage of the present invention is:
1. adopt liquid at high speed to cultivate and solid expansion bonded fermentation technique, both guaranteed the high-speed rapid growth of probiotics, guaranteed that again its meta-bolites keeps fully;
2. the high density lactic acid in the product can significantly improve the digestion and absorption function of aquatic animal, and produced simultaneously peptidoglycan has increased the immunological competence of aquatic animal to pathogenic agent;
3. the yeast of product middle and high concentration can improve the abundant single cell protein that the fish bulk-growth needs, and simultaneously, yeast cell is discharged a large amount of β-1,3 dextran by abundant broken wall, significantly improves the immunological competence to Gram-positive and negative bacterium;
4. the nutritious solid medium of milk-acid bacteria and saccharomycetes to make fermentation produces a large amount of cellulases, hemicellulase, oligopeptide, small peptide mixture, microbiotic, trace element and other meta-bolites, for aquatic animal provides the best condition of nourishing and growing;
5. adopt food crop and common feedstuffs carrier as the main medium raw material, reduced production cost, use fermentation and production unit commonly used simultaneously, be easy in aquaculture, promote;
6. product of the present invention adds in the aquatic feeds, provide abundant milk-acid bacteria to improve the ability of digesting and assimilating of animal, provide abundant microbial polysaccharide to improve the non-specific immunity of culturing body, reduced the usage quantity of microbiotic in breed, not having any microbiotic and heavy-metal residual in animal body, is a kind of safe, green microniological proudcts.
Embodiment
The invention will be further described below in conjunction with embodiment.
Embodiment 1
The first step: fermentation strain seed selection
A. bacterial strain screening: extracting secretory product in the crucian enteron aisle of health is the bacterium source, adopts MRS milk-acid bacteria substratum to carry out milk-acid bacteria and separates, and filters out plant lactobacillus, selects substratum to separate S. cervisiae with yeast in beer fermentation liquid; Isolating plant lactobacillus, S. cervisiae are preserved bacterial classification respectively under 4 ℃ in yeast extract paste wort agar substratum; What the present invention used is that the following bacterial classification that screens is:
Lactobacillus plantarum (ATCC8014) plant lactobacillus, Saccharomycscerevisiac (IFO0203) S. cervisiae,
B. be inoculated in isolating plant lactobacillus, S. cervisiae in the broth culture respectively, after cultivating 22 hours under 37 ℃ of temperature, S. cervisiae is inoculated in yeast extract paste wort agar substratum, plant lactobacillus is inoculated in the milk-acid bacteria substratum, carry out streak culturely, select that growth is fast, thalline evident characteristic plant lactobacillus, S. cervisiae preserve respectively in the laboratory;
C. the production production of hybrid seeds: will select the fast plant lactobacillus of growth at last and transfer in the eggplant bottle that nutrient agar is housed, S. cervisiae is transferred and the yeast extract paste wort agar is housed does in the eggplant bottle of substratum, under 37 ℃ of temperature, cultivated 47 hours, band eggplant bottle surface lawn is covered with, and can take out when band is yellow in white, put into 4 ℃ refrigerator then and preserve;
MRS milk-acid bacteria substratum: Yeast extract (yeast extract paste) 7.5g Peptone (peptone) 7.5Glucose (glucose) 10g, KH
2PO
42g (potassium primary phosphate), Tomato juice (Tomato juice) 100ml Tween (tween) 80 0.5ml, Distilled water (distilled water) 900mlpH 7.0;
Yeast selects substratum to be: glucose 1g, KCl1.8g, yeast extract 2.5g, sodium-acetate g.2g, agar 15-20g, distilled water 1000ml.
Nutrient agar: extractum carnis 3g, peptone 10g, sodium-chlor 5g, agar 15-20g, water 1000ml, pH7.0-7.2;
Broth culture: extractum carnis 3g, peptone 10g, sodium-chlor 5g, water 1000ml, pH7.0-7.2, yeast extract paste wort agar substratum: malt meal 3g, yeast extract 0.1g, water 1000ml.
More than several substratum all on the books on laboratory operation is taught book;
Second step: the liquid culture of useful bacterial strain
A. take by weighing each bacterial classification by following weight proportion: plant lactobacillus 55%, S. cervisiae 45%;
B. the plant lactobacillus that takes by weighing and S. cervisiae are mixed and carry out compound cultivation: the mixing lawn that takes by weighing among the step a is inoculated in 121 ℃, the liquid nutrient medium of 45min high-temperature sterilization in 1: 25 ratio, be poured on and stir 30min in the fermentor tank, its rotating speed is 220r/min, putting into sterilization back plastic tank after stirring left standstill 5 days, leave standstill the gas of measuring Ph value and release output in the process every day
C. sampling 3 times in the fermenting process, microscopically detects the thalli growth situation and measures Ph value, stops to ferment when reaching 3.0-4.0 with the Ph value when total viable count reaches 2,000,000,000/ml concentration more than;
Liquid nutrient medium: dregs of beans 2%, urea 1%, ammonium sulfate 0.5%, yeast powder 1%, Semen Maydis powder 3%, molasses 10%, potassium primary phosphate 0.5%, SODIUM PHOSPHATE, MONOBASIC 0.7%, trimagnesium phosphate 0.6%, sal epsom 0.2%, sterilized water 80.5%;
The 3rd step: centrifugal collection somatic cells wall
B. with fermented liquid in the step 1 at the outsourcing whizzer with every 1kg8000 r/min, the centrifugal collection bacterial sediment of 10min, extremely white with 0.9% physiological saline repetitive scrubbing bacterium, thalline is suspended in the distilled water, handle 30-40min with outsourcing ultrasonic cell-break machine then, carry out physics broken 2-4 time;
C. cell walls separates: with the solution after the physics fragmentation at whizzer with the centrifugal 15-20min of every 1kg4000r/min, remove throw out, to remove not broken thalline, again with supernatant at whizzer with the centrifugal 20min of every 1kg8000r/min, the thickness cell wall of collecting precipitation;
The 4th step: the solid enlarged culturing of flora
A. the bacterium liquid of the compound cultivation of step 2 is mixed in the solid enlarged culturing base of 121 ℃ of ratio accesses by 1: 25,30min high-temperature sterilization, stir 30min to even, pour the 50kg sealing and fermenting in the double-deck feedbag of sterilizing into, 30 ℃ of temperature, time 8-10 days, sampling was three times in the fermenting process, measures moisture and Ph value, take a sample after the fermentation ends, mensuration moisture is that 35-40%, Ph value are 3.5-4.0;
B. add the thickness cell wall: cell walls gleanings and solid are enlarged fermented product by 1: 50 mixed, be poured in the stirrer and stir, rotating speed is 200 commentaries on classics/min, and churning time is 30min;
Solid enlarged culturing base: dregs of beans 2%, molasses 8%, Semen Maydis powder 4%, clear chaff 40%, potassium primary phosphate 0.03%, SODIUM PHOSPHATE, MONOBASIC 1%, sal epsom 0.4%, urea 1%, sterilized water 43.57%;
The 5th step: pulverize and add special carrier
A. dry: the product of step 4 fermentation finished thoroughly1 is in chronological sequence put into the incubator drying, every dish weight 2.0 kilograms ± 0.5, temperature is controlled at 40-45 ℃, and time 14-16 hour, the material moisture content that drying is finished is controlled at≤and 12%;
B. add carrier; Dried product is added carrier in 1: 0.02 ratio, and carrier is formed and ratio is zeolite powder 40%, lime carbonate 60%, pours in the stirrer to stir; Change between pulverizing and pulverize, temperature of charge is controlled at below 45 ℃, and the material after the pulverizing is crossed 50 eye mesh screens, and sieve back coarse fodder is pulverized again;
C. packing into after sieving has in the bucket or bag of inner bag, tightens sack, fastens Sign Board, changes stockyard over to and stacks in order; Sampling detects, significant parameter: moisture content≤12%, viable count 60-80 hundred million/g; Physical behavior: deep yellow pulverulent solids.
Embodiment 2
The first step: fermentation strain seed selection: with embodiment 1;
Second step: the liquid culture of useful bacterial strain
A. take by weighing each bacterial classification by following weight proportion: plant lactobacillus 65%, S. cervisiae 35%
B. the plant lactobacillus that takes by weighing and S. cervisiae are mixed and carry out compound cultivation: the mixing lawn that takes by weighing among the step a is inoculated in 121 ℃, the substratum of 45min high-temperature sterilization in 1: 30 ratio, in fermentor tank, stir 30min, its rotating speed is 200r/min, put into sterilization back plastic tank after stirring and left standstill 7 days, leave standstill the gas of measuring Ph value and release output in the process every day;
C. sampling 3 times in the fermenting process, microscopically detects the thalli growth situation and measures Ph value, stops to ferment when 2,000,000,000/ml concentration and Ph value reach 3.0-4.0 when total viable count reaches;
Liquid nutrient medium: dregs of beans 6%, urea 3%, ammonium sulfate 1%, yeast powder 3%, Semen Maydis powder 8%, molasses 20%, potassium primary phosphate 1%, SODIUM PHOSPHATE, MONOBASIC 1.9%, trimagnesium phosphate 0.8%, sal epsom 0.9%, sterilized water 54.4%;
The 3rd step: centrifugal collection somatic cells wall
A. with fermented liquid in the step 1 at the outsourcing whizzer with every 1kg8000 r/min, the centrifugal collection bacterial sediment of 10min, extremely white with 0.9% physiological saline repetitive scrubbing bacterium, thalline is suspended in the distilled water, handle 30-40min with outsourcing ultrasonic cell-break machine then, carry out physics broken 2-4 time;
B. cell walls separates: with the solution after the physics fragmentation at whizzer with the centrifugal 15-20min of every 1kg4000r/min, remove throw out, to remove not broken thalline, again with supernatant at whizzer with the centrifugal 20min of every 1kg8000r/min, the thickness cell wall of collecting precipitation;
The 4th step: the solid enlarged culturing of flora
A. the bacterium liquid of the compound cultivation of step 2 is mixed in the solid enlarged culturing base of 121 ℃ of ratio accesses by 1: 30,45min high-temperature sterilization, stir 45min to even, pour the 70kg sealing and fermenting in the double-deck feedbag of sterilizing into, 35 ℃ of temperature, 10 days time, sampling is three times in the fermenting process, measures moisture and Ph value, take a sample after the fermentation ends, mensuration moisture is that 35-40%, Ph value are 3.5-4.0
B. add the thickness cell wall: cell walls gleanings and solid are enlarged fermented product by 1: 60 mixed, pour in the stirrer and stir, rotating speed is 180 commentaries on classics/min, and churning time is 30min;
Solid enlarged culturing base: bean cake powder 6%, molasses 12%, Semen Maydis powder 8%, clear chaff 55%, potassium primary phosphate 0.06%, SODIUM PHOSPHATE, MONOBASIC 2%, sal epsom 0.8%, urea 1.8%, sterilized water 43.3%
The 5th step: pulverize and add special carrier
A. dry: the product of step 4 fermentation finished thoroughly1 is in chronological sequence put into the incubator drying, and temperature is controlled at 45 ℃, 16 hours time, and the material moisture content that drying is finished is controlled at≤and 12%;
B. add carrier; Dried product is added carrier in 1: 0.03 ratio, and carrier is formed and ratio is zeolite powder 50%, lime carbonate 50%, pours in the stirrer to stir; Change between pulverizing and pulverize, temperature of charge is controlled at below 45 ℃, and the material after the pulverizing is crossed 50 eye mesh screens, and sieve back coarse fodder is pulverized again;
All the other are the same with embodiment 1.
Add in 1-1.5 ‰ ratio during use and throw something and feed after aquatic feeds stirs, between the aquatic animal period of disease, should strengthen usage quantity, in 2-3 ‰ ratio add use disappear to disease symptom till, this product needs to seal up for safekeeping in that dry ventilation is airtight, should use as early as possible behind the Kaifeng.Under the leader's of the Shanghai City State Scientific and Technological Commission care and support, carry out the three-dimensional health of freshwater shrimp in beginning in 2002 in the Qingpu District, Shanghai City and culture research, from experimental data as can be seen, the per mu yield on test each pool, the pool is 151.8 jin, and the mean yield on the contrast pool only is 70 jin, the output on the test pool has improved 116.9%, and difference is quite obvious, and feed coefficient has on average reduced by 17.1%.The average disease in the contrast pool is 1.8 times in culture-cycle, and tests the average disease in the pool 0.5 time.And the individuality of respectively testing pool freshwater shrimp is bigger, and body surface smooth, body colour are blue or green, selling price is higher, and each pool freshwater shrimp body surface of the contrast pool is crude, drift is more, and body colour is whiter, and selling price is not high.
Claims (4)
1. a preparation method who utilizes lactobacillus and yeast to produce microbe polysaccharides formulation is characterized in that,
Adopt liquid at high speed to cultivate and solid expansion bonded fermentation process, its production stage is:
The first step: fermentation strain seed selection
A. bacterial strain screening: with secretory product in the crucian enteron aisle of health is the bacterium source, adopts MRS milk-acid bacteria substratum to carry out milk-acid bacteria and separates, and filters out a kind of plant lactobacillus, selects substratum to separate S. cervisiae with yeast in beer fermentation liquid; Isolating two kinds of bacterial classifications are placed on respectively in the yeast extract paste wort agar substratum, under 4-6 ℃ of temperature, preserve bacterial classification;
B. be inoculated in isolating plant lactobacillus and S. cervisiae in the broth culture respectively, under 35-40 ℃ of temperature, cultivated 20-25 hour, then S. cervisiae is inoculated in yeast extract paste wort agar substratum, plant lactobacillus is inoculated in the milk-acid bacteria substratum, carry out streak culturely, select that growth is fast, thalline evident characteristic plant lactobacillus, S. cervisiae preserve respectively in the laboratory;
C. the production production of hybrid seeds: will select the fast plant lactobacillus of growth at last and transfer in the eggplant bottle that nutrient agar is housed, the S. cervisiae yeast extract paste wort agar of transferring is done in the eggplant bottle of substratum, under 35-40 ℃ of temperature, cultivated 45-50 hour, treat that eggplant bottle surface lawn is covered with, and can take out when band is yellow in white, put into 2-6 ℃ refrigerator then and preserve;
Second step: the liquid culture of useful bacterial strain
A. take by weighing each bacterial classification by following weight proportion example: plant lactobacillus 55-65%, S. cervisiae 35-45%;
B. the plant lactobacillus that takes by weighing and S. cervisiae are mixed and carry out compound cultivation: the mixing lawn that takes by weighing among the step a is in 1: the ratio of 25-30 is inoculated in the liquid nutrient medium of 120-130 ℃ of high-temperature sterilization, in fermentor tank, stir 30min, its rotating speed is 200-240r/min, put into sterilization back plastic tank after stirring and left standstill 5-7 days, leave standstill the gas of measuring Ph value and release output in the process every day;
C. sampling 3 times in the fermenting process, microscopically detects the thalli growth situation and measures Ph value, stops to ferment when 2,000,000,000/ml concentration and Ph value reach 3.0-4.0 when total viable count reaches;
The 3rd step: centrifugal collection somatic cells wall
A. with fermented liquid in the step 1 at whizzer with every 1kg8000r/min, the centrifugal collection bacterial sediment of 10min, is suspended in thalline in the distilled water to white with 0.9% physiological saline repetitive scrubbing bacterium, handle 30-40min with the ultrasonic cell-break machine then, carry out physics broken 2-4 time;
B. cell walls separates: with the solution after the physics fragmentation at whizzer with the centrifugal 15-20min of every 1kg4000r/min, remove throw out, to remove not broken thalline, again with supernatant at whizzer with the centrifugal 20min of every 1kg8000r/min, the thickness cell wall of collecting precipitation;
The 4th step: the solid enlarged culturing of flora
A. the bacterium liquid with the compound cultivation of step 2 mixes in 1: in 120-130 ℃ of the ratio access of 25-30, the solid enlarged culturing base of 30-45min high-temperature sterilization, stir 30-45min to even, to going into the 50-70kg sealing and fermenting in the double-deck feedbag of sterilizing, temperature 30-35 ℃ time 8-10 days, sampling is three times in the fermenting process, measure moisture and Ph value, take a sample after the fermentation ends, mensuration moisture is that 35-40%, Ph value are 3.5-4.0;
B. add the thickness cell wall: cell walls gleanings and solid are enlarged fermented product by 1: the 50-60 mixed, be poured in the stirrer and stir, rotating speed is 150-200 commentaries on classics/min, churning time is 30min;
The 5th step: pulverize and add special carrier
A. dry: the product of step 4 fermentation finished thoroughly1 is in chronological sequence put into the incubator drying, and temperature is controlled at the 40-45 degree, and time 14-16 hour, the material moisture content that drying is finished is controlled at≤and 12%;
B. add carrier; With dried product in 1: the ratio of 0.02-0.03 is added carrier, pours in the stirrer to stir, and changes pulverizer then over to and pulverizes, and temperature of charge is controlled at below 45 ℃, and the material after the pulverizing is crossed 50 eye mesh screens, and sieve back coarse fodder is pulverized again;
C. packing into after sieving has in the bucket or bag of inner bag, tightens sack, fastens Sign Board, changes stockyard over to and stacks in order; Sampling detects, significant parameter: moisture content≤12%, viable count 60-80 hundred million/g; Physical behavior: deep yellow pulverulent solids.
2. the preparation method who utilizes lactobacillus and yeast to produce microbe polysaccharides formulation according to claim 1, it is characterized in that, described step 2 liquid nutrient medium is that sterilized water adds bean cake powder again, urea, ammonium sulfate, yeast powder, Semen Maydis powder, molasses, potassium primary phosphate, Sodium phosphate dibasic, trimagnesium phosphate, at least three kinds of mixtures of sal epsom, its weight percent proportioning is: dregs of beans 2-6%, urea 1-3%, ammonium sulfate 0-1%, yeast powder 0-3%, Semen Maydis powder 2-8%, molasses 10-22%, potassium primary phosphate 0-1%, SODIUM PHOSPHATE, MONOBASIC 0-2%, trimagnesium phosphate 0-1%, sal epsom 0-1%, sterilized water 54-84%.
3. the preparation method who utilizes lactobacillus and yeast to produce microbe polysaccharides formulation according to claim 1, it is characterized in that, the solid enlarged culturing base of described step 4 is that clear chaff and sterilized water add bean cake powder again, molasses, Semen Maydis powder, potassium primary phosphate, SODIUM PHOSPHATE, MONOBASIC, sal epsom, at least four kinds of mixtures in the urea are formed, and its weight percent proportioning is: dregs of beans 2-6%, molasses 8-12%, Semen Maydis powder 4-8%, clear chaff 40-70%, potassium primary phosphate 0.03-0.06%, SODIUM PHOSPHATE, MONOBASIC 1-2%, sal epsom 0.4-0.8%, urea 1-2%, sterilized water 15-45%.
4. the preparation method who utilizes lactobacillus and yeast to produce microbe polysaccharides formulation according to claim 1, it is characterized in that, the special carrier of described step 4 is made up of zeolite powder, lime carbonate, and its weight percent proportioning is: zeolite powder 40-50%, lime carbonate 50-60%.
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| CN101715877A (en) * | 2009-12-14 | 2010-06-02 | 上海恩创生物技术研究所 | Method for preparing functional microbial preparation from lactic acid bacillus and yeast |
| CN101712987B (en) * | 2009-09-21 | 2012-01-25 | 内蒙古农业大学 | Method for quickly, qualitatively and quantitatively measuring Lactobacillus plantarum in probiotic dairy products |
| CN103396496A (en) * | 2013-07-16 | 2013-11-20 | 宁波大学 | Preparation method of modified lactobacillus acidophilus peptidoglycan |
| CN103404716A (en) * | 2013-07-09 | 2013-11-27 | 青岛中浩环能技术开发有限公司 | Compound microorganism bacterium powder and preparation method thereof |
| CN101671635B (en) * | 2009-09-21 | 2013-12-25 | 通化万赢生物科技有限公司 | Compound microbial agent and preparation method thereof |
| CN104152370A (en) * | 2014-05-13 | 2014-11-19 | 江苏绿华生物工程有限公司 | Culture medium and method for mixed culture of streptococcus lactis, lactobacillus plantarum and flavour-producing yeast |
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| CN104450582A (en) * | 2014-12-10 | 2015-03-25 | 北京世纪新探生物科技有限公司 | Lactobacillus plantarum, feed premix containing lactobacillus plantarum and preparation method of lactobacillus plantarum |
| CN105483050A (en) * | 2015-12-31 | 2016-04-13 | 北京英惠尔生物技术有限公司 | Lactobacillus plantarum strain and application thereof in fermented feed |
| CN110526498A (en) * | 2019-08-23 | 2019-12-03 | 齐鲁理工学院 | A kind of method of lactic acid bacteria processing sanitary sewage |
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| CN101671635B (en) * | 2009-09-21 | 2013-12-25 | 通化万赢生物科技有限公司 | Compound microbial agent and preparation method thereof |
| CN101712987B (en) * | 2009-09-21 | 2012-01-25 | 内蒙古农业大学 | Method for quickly, qualitatively and quantitatively measuring Lactobacillus plantarum in probiotic dairy products |
| CN101715877A (en) * | 2009-12-14 | 2010-06-02 | 上海恩创生物技术研究所 | Method for preparing functional microbial preparation from lactic acid bacillus and yeast |
| CN104379001A (en) * | 2012-06-25 | 2015-02-25 | 戴姆有限公司 | Fat binder obtained from biomass resulting from beer production |
| CN103404716A (en) * | 2013-07-09 | 2013-11-27 | 青岛中浩环能技术开发有限公司 | Compound microorganism bacterium powder and preparation method thereof |
| 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 |
| CN103396496A (en) * | 2013-07-16 | 2013-11-20 | 宁波大学 | Preparation method of modified lactobacillus acidophilus peptidoglycan |
| CN103396496B (en) * | 2013-07-16 | 2015-07-08 | 宁波大学 | Preparation method of modified lactobacillus acidophilus peptidoglycan |
| CN104152370A (en) * | 2014-05-13 | 2014-11-19 | 江苏绿华生物工程有限公司 | Culture medium and method for mixed culture of streptococcus lactis, lactobacillus plantarum and flavour-producing yeast |
| CN104450582A (en) * | 2014-12-10 | 2015-03-25 | 北京世纪新探生物科技有限公司 | Lactobacillus plantarum, feed premix containing lactobacillus plantarum and preparation method of lactobacillus plantarum |
| CN105483050A (en) * | 2015-12-31 | 2016-04-13 | 北京英惠尔生物技术有限公司 | Lactobacillus plantarum strain and application thereof in fermented feed |
| CN105483050B (en) * | 2015-12-31 | 2018-12-18 | 北京英惠尔生物技术有限公司 | One lactobacillus plantarum and its application in fermented feed |
| CN110526498A (en) * | 2019-08-23 | 2019-12-03 | 齐鲁理工学院 | A kind of method of lactic acid bacteria processing sanitary sewage |
| CN114601174A (en) * | 2020-12-04 | 2022-06-10 | 安琪纽特股份有限公司 | Lactobacillus lysate and preparation method and application thereof |
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