CN101186892A - Breeding for oligo-acid production bacterium and method for producing oligo-acid thereof - Google Patents
Breeding for oligo-acid production bacterium and method for producing oligo-acid thereof Download PDFInfo
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- CN101186892A CN101186892A CNA2007100505735A CN200710050573A CN101186892A CN 101186892 A CN101186892 A CN 101186892A CN A2007100505735 A CNA2007100505735 A CN A2007100505735A CN 200710050573 A CN200710050573 A CN 200710050573A CN 101186892 A CN101186892 A CN 101186892A
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- sodium alginate
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Abstract
The invention discloses a method for obtaining algalase and producing oligomeric acid (or namely oligomeric alginic acid) by culturing microorganisms. Microorganism brevibacterium is obtained by being extracted from tidal flat mangrove sludge, the method of inducing enzyme production and fermentation after strains are obtained is that moderate sodium alginate, nitrogen source and inorganic salt are employed to form solution which is cultured at ordinary temperature for 36 to 48 hours, and then a medicament of the algalase of high activity is obtained. The method of enzymolysis for sugar production is that enzyme solution and sodium alginate solution are mixed according to a certain proportion for processing enzymolysis reaction under quite mild conditions, the sodium alginate can be transferred to oligomeri acid in a short time, and the product is obtained by concentrating or drying. The strains of the method has the advantages of being stable in heritability, high in yielding enzyme, rapid in fermentation and enzymolysis and normalized in conditions of fermentation and enzymolysis, and the quality of the product is good.
Description
Technical field
The present invention relates to the production method of oligomerization acid, particularly utilize the method for microorganisms producing oligomerization acid.
Technical background
Lalgine is the polyose carbohydrate that for example extracts the sea-tangle from the phaeophyta plant.The Lalgine molecule is by β-D-1,4-mannuronic acid and α-L-1, and the block linear polymer that two kinds of monomers of 4-guluronic acid are formed, molecular weight is about 106.Various biosynthetic Lalgine structures are similar substantially.But two kinds of uronic acids forming Lalgine are because of biological different variant.In a molecule, may only contain the continuous segment that wherein a kind of uronic acid constitutes, also may constitute segmented copolymer by two kinds of uronic acid chain links.
In recent years, Lalgine is because its purposes and by people's pay attention to day by day widely.In foodstuffs industry, Lalgine is used to make artificial foods such as artificial fruit, artificial egg, jelly, also is used as frostproofer, anti-settling agent, school flavor agent and the ice-creams etc. of food thickener, beverage, is referred to as marvellous foodstuff additive by people.Aspect preventing and curing diseases, Lalgine has effects such as the intravital cholesterol level of the people of reduction, dredging vascellum, blood viscosity lowering, vessel softening, is described as health long-life food by people.In textile industry, printing and dyeing figured cloth, starching all need to use in a large number Lalgine, up to the present, also can not replace the better material of Lalgine.Aspect light industry, utilize fixing various bacterial classifications of Lalgine and zymin, thereby continuously ferment, enzymolysis boosts productivity greatly.In addition, Lalgine is also as capsule, with its safely, advantage such as have no side effect is used in the pharmaceutical industry.On fodder industry, Lalgine also is used as binding agent, phagostimulant.
On agricultural, seaweed fertilizer is to be main raw material with the natural seaweed, handle through special biochemical process, a kind of natural organic fertilizer that extracts the elite material in the marine alga and obtain, it has greatly kept the natural radioactivity component in the marine alga, contain a large amount of non-itrogenous organic substances and the incomparable potassium of terrestrial plant, calcium, magnesium, the mineral element of kind and abundant VITAMIN surplus the iron etc. 40, contain peculiar Sargassum polysaccharides in the marine alga especially, alginic acid, highly unsaturated fatty acids and multiple natural phant growth regulator, has very high biological activity, but the generation of non-specific active factor in the stimulating plant body, the balance of regulating endogenous hormones.
Influencing production efficiency owing to Lalgine viscosity is high is that present Lalgine is produced a big problem that exists in the solid seaweed fertilizer.The exploitation of oligomerization acid biotechnology not only can solve the full-bodied problem of Lalgine, also solves the non-full nutrition problem that contains the Lalgine that the 10-20% plant can not utilize in the seaweed fertilizer product simultaneously.Oligomerization acid is oligo-alginate, with oligomerization acid is that the seaweed organism fertilizer of foundation development extracts from sargassun, can regulate crop growth, increase yield and quality, reduce disease and pest, strengthening a class efficient biologic-organic fertilizer of crop anti-adversity, is the pure natural plants growth stimulant, to environment and crop itself without any side effect and pollution.
Open source literature has been reported the preparation of some alginases, mainly is process screening from some microorganisms, and selecting those can be raw material with the sodium alginate, and culture condition is uncomplicated, can obtain the bacterial classification of high reactivity alginase again.The Erichsen that discloses through screening as " Chinese Marine University's journal " the 2nd phase in 2006 replaces Zymomonas mobilis as the bacterial classification that produces alginase, and culture condition comprises temperature, pH value, time, optimum carbon source, nitrogenous source to producing the influence of enzyme, and its enzyme activity generally can reach about 0.18u/mL." ocean science " 2006 the 2nd phases are with the bacterial classification of other zygosaccharomyces as the generation alginase, its two strain bacterial strains that filter out have been carried out the analysis of alginase characteristic in sodium alginate degradation capability and the extracellular products respectively, and resulting alginase vigor reaches as high as 0.36u/mL.
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing the acid of microorganisms producing oligomerization, particularly can produce the marine microorganism of high reactivity alginase and utilize the high reactivity alginase of this microorganism and its generation that sodium alginate is degraded, make it to obtain the production method of the water-soluble oligomerization acid of excellent performance.
The inventor is engaged in the research and development of marine biotechnology technical matters for many years always, and speciality is in the application and development and the test and analysis technology research in Sargassum polysaccharides field.Influencing production efficiency owing to Lalgine viscosity is high is that present solid seaweed fertilizer is produced a big problem that exists.The exploitation of oligomerization acid biotechnology not only can solve the full-bodied problem of Lalgine, also solves the non-full nutrition problem that contains the Lalgine that the 10-20% plant can not utilize in the seaweed fertilizer product simultaneously.
The inventor can grow in the substratum that with the sodium alginate is sole carbon source in the strain that mountain pass, Beihai, Guangxi Zhuang Autonomous Region mangrove forest area is found, justacrine goes out the strain excellent of alginase.
Through the above-mentioned strain excellent that screens is cultivated in inducing culture, its inductor is a sodium alginate, finds that its oozy alginase has higher activity and very stable heredity, and it belongs to brevibacterium sp preliminary evaluation.Can under 4 ℃ or normal temperature, preserve.
The applicant has delivered to this bacterial classification Chinese typical culture collection center preservation at present, and preservation date is on October 30th, 2007, and deposit number is CCTCC M 207168, the classification called after brevibacterium sp of suggestion.
The surface characteristic of this bacterium is: Gram-positive.Grow on beef-protein medium, the color of bacterium colony is a milk yellow.
The production technique of the spawn culture of alginic-acid-decomposing bacteria of the present invention and oligomerization acid is as follows:
Be the alginic-acid-decomposing bacteria enlarged culturing of CCTCC M 207168 with China's typical culture collection center preserving number earlier, prepare oligomerization acid with two-step approach again: the first step is used and is induced the product enzymic fermentation, preparation crude liquid type alginase preparation; The crude liquid type alginase preparation that second step used the first step to produce is degraded to alginic acid solution, the acid of preparation oligomerization.
The enlarged culturing of above-mentioned said alginic-acid-decomposing bacteria is that the bacterial classification of screening is cultivated in medium, and the medium of cultivation can be solid medium or liquid nutrient medium.Described solid medium is that sodium alginate is selected substratum, wherein the weight percentage of each composition is: sodium alginate 0.5-2%, ammonium sulfate 0.5-1%, potassium primary phosphate or dipotassium hydrogen phosphate 0.05-0.1%, SODIUM PHOSPHATE, MONOBASIC or Sodium phosphate dibasic 0.05-0.1%, sal epsom, ferrous sulfate, each 0.01-0.1% of calcium chloride;
The weight percentage of described each composition of liquid nutrient medium is: sodium alginate 0.5-2%, urea 0.5-1%, potassium primary phosphate or dipotassium hydrogen phosphate 0.05-0.1%, SODIUM PHOSPHATE, MONOBASIC or Sodium phosphate dibasic 0.05-0.1%, sal epsom, ferrous sulfate, each 0.01-0.1% of calcium chloride; Or make solvent with seawater, and adding sodium alginate 0.5-2%, urea or other nitrogenous source 0.5-1%, and molysite, each 0.01-0.1% of calcium salt regulate the pH value to 6.0-8.0.
The temperature of cultivating at the enlarged culturing base is 25-35 ℃, and the time is 1-3 days.
The bacterial classification that obtains from enlarged culturing is reserved seed for planting the refrigerator that is stored in 4 ℃ on a small quantity, and major part can be directly used in industrial fermentation and produce the alginase preparation.The equipment of suitability for industrialized production alginase preparation adopts the fermentor tank of belt stirrer and heating unit, production process at first is that product enzymic fermentation substratum is induced in preparation, method is on the basis of sodium alginate substratum, add the short enzyme releasing hormone of 0.5-1% peptone or other nitrogen source and 0.01-0.1% again, the bacterial classification that adds enlarged culturing then, add the 0.5-1% of the amount of bacterial classification by total sodium alginate total amount, regulate pH6.0-8.0, the best is 6.5-7.0, temperature keeps 25-35 ℃, fermentation time 36-48 hour, fermenting process is regularly analyzed material, general when gemma occurring in the fermented liquid, the alginase vigor in the fermented liquid can finish fermentation when peaking.Fermented liquid after the centrifugal slagging-off degerming is crude liquid type alginase preparation.
With this crude enzyme liquid and 2%-6% sodium alginate soln by a certain percentage (as 1: 10-1: 20V/V) mixing, at 40 ℃-50 ℃, carry out alignic enzyme digestion reaction under the pH6.0-7.0 condition.Because the molecular weight difference of substrate sodium alginate, the asynchronism(-nization) of enzyme digestion reaction terminated generally is no more than 3-12 hour, and sodium alginate promptly all is converted into water-soluble oligomerization acid.Compare and the enzyme digestion reaction time at the bottom of utilizing different enzymes, can obtain the oligomerization acid product of different molecular weight distribution range.
The enzyme digestion reaction mixture carries out 50-70 ℃ the high temperature enzyme that goes out lives, and concentrates and purifying through ultrafiltration, nanofiltration, and spraying drying obtains the oligomerization acid product, and this product can be used as pharmacy, agricultural chemicals, food or light industry raw material etc.
Compare with the technology of present disclosed document, the present invention has outstanding substantive distinguishing features and progress is:
1. the bacterial classification that obtains from beach mangrove forest mud is a tyrothricin, can adopt general method of breeding strain, and process is uncomplicated.Its heredity is very stable; Enzymatic productivity does not descend after going down to posterity 5 years, preserves above non-inactivation half a year at room temperature 15-40 ℃ condition lower seal, and enzymatic productivity does not descend yet.
2. produce enzymic fermentation culture medium culturing bacterial classification of the present invention with inducing, its enzymatic productivity height, the contained alginase vigor of unit fermented liquid height, and can suppress the growth of assorted bacterium, and alginic acid consumption is few, and culture condition is controlled easily, the alginase preparation is active high, enzyme digestion reaction is very gentle, and the short period of time can be converted into alginic acid oligomerization acid, and concentrated or drying obtains product.
3. the time of producing enzymic fermentation and enzyme digestion reaction is short, can finish the whole process of fermentation and enzymolysis in general 40-48 hour, very favourable to suitability for industrialized production, show and to enhance productivity at short notice, the bacterial classification genetic stability, the zymogenic rate height ferments and enzymatic hydrolysis condition routinizes, speed is fast, good product quality saves production cost.
4. the enzyme digestion reaction mild condition is easy to control reaction conditions to obtain the oligomerization acid product of different molecular weight distribution range.
Below be embodiments of the invention:
Embodiment one
1. liquid nutrient medium enlarges bacterial classification.Be that the tyrothricin bacterial classification of CCTCC M 2,071 68 inserts the liquid sodium alginate from slant medium and selects the substratum with deposit number, sodium alginate 0.5-2%, ammonium sulfate 0.5-1%, potassium primary phosphate or dipotassium hydrogen phosphate 0.05-0.1%, SODIUM PHOSPHATE, MONOBASIC or Sodium phosphate dibasic 0.05-0.1%, sal epsom, ferrous sulfate, each 0.01-0.1% of calcium chloride; Or add the 0.5-1% peptone on its basis, regulate the pH value to 6.0-8.0.The temperature of cultivating at the enlarged culturing base is 25-35 ℃, and the time is 1-3 days.
2. produce enzymic fermentation production.Above-mentioned expansion bacteria culture fluid 300mL is added the 600L fermentor tank, and (jar is interior by above-mentioned product enzymic fermentation substratum 400L, short enzyme releasing hormone 0.01-0.1%, regulate pH6.0-8.0, temperature 25-35 ℃, fermentation time 42 hours, the alginase activity peaks in the fermented liquid at this moment, stops fermentation, and centrifugal then slagging-off degerming promptly gets crude liquid type alginase preparation.
3. enzymolysis produces sugar production.In the 5000L reactor, with above-mentioned crude enzyme liquid and 1: 10 volume ratio mixing of 2-6% sodium alginate soln ammonium, 40 ℃-50 ℃, reaction is 6 hours under the pH6.0-7.0 condition, is warming up to 60 ℃ of enzymes that go out and lives, and spraying drying obtains rough oligomerization acid pulvis behind 10 times of the ultrafiltration and concentration.
Embodiment two
1. solid medium is cultivated bacterial classification.The substratum that preparation is made up of sodium alginate, urea and inorganic salt, wherein the weight percentage of each composition is: sodium alginate 0.5-2%, ammonium sulfate 0.5-1%, potassium primary phosphate or dipotassium hydrogen phosphate 0.05-0.1%, SODIUM PHOSPHATE, MONOBASIC or Sodium phosphate dibasic 0.05-0.1%, sal epsom, ferrous sulfate, each 0.01-0.1% of calcium chloride.Bacterial classification is inserted on this substratum, cultivated 3-5 days, and obtained enlarging bacterial classification for 25-30 ℃.
2. produce enzymic fermentation production.Above-mentioned expansion bacterial classification is added to for 50 kilograms to be placed with in advance produces the enzymic fermentation substratum and (in jar above-mentioned product enzymic fermentation substratum 400L is arranged, short enzyme releasing hormone 0.01-0.1%) in 4 tons of fermentor tank, regulate pH6.0-8.0, temperature 25-35 ℃, fermentation time 42 hours, the alginase activity peaks in the fermented liquid at this moment, stops fermentation, and centrifugal slagging-off degerming promptly gets crude liquid type alginase preparation.
3. enzymolysis produces sugar production.Method by embodiment one is carried out.
Embodiment three
1. liquid nutrient medium enlarges bacterial classification.Slant strains inserted be placed with in advance in the 200L kind jar of embodiment-sodium alginate substratum, 25-30 ℃ of aerated culture 24 hours obtains liquid and enlarges bacterial classification.
2. produce enzymic fermentation production.Above-mentioned expansion bacterial classification is added to is placed with 3 tons in advance and produces the enzymic fermentation substratum and (in jar above-mentioned product enzymic fermentation substratum 400L is arranged, short enzyme releasing hormone 0.01-0.1%) in the fermentor tank, regulate pH6.0-8.0, temperature 25-35 ℃, fermentation time 42 hours, centrifugal slagging-off degerming promptly get crude liquid type alginase preparation.
3. enzymolysis produces sugar production.Method by embodiment one is carried out.
Embodiment four
1. solid medium enlarges bacterial classification.The sodium alginate substratum is paved into the inclined-plane in the eggplant-shape bottle of 500mL, bacterial classification is inserted the eggplant-shape bottle inclined-plane by the test tube slant, cultivated 3-5 days, and obtained enlarging bacterial classification for 25-30 ℃.
2. produce enzymic fermentation production.The expansion bacterial classification of an above-mentioned eggplant-shape bottle is had access to 400L induce in the 0.6 ton of fermentor tank that produces the enzyme substratum, pH6.0-8.0, temperature 25-35 ℃, fermentation time stops after 42 hours fermenting.Centrifugal slagging-off degerming obtains the comparatively liquid-type alginase preparation of purifying behind ammonium sulfate precipitation, dialysis desalination.
3. enzymolysis produces sugar production.With above-mentioned enzyme liquid and 3% purified alginate solution mixing, 40 ℃-50 ℃, enzymolysis is 4 hours under the pH6.0-7.0 condition, adjust pH to 2.0 termination reaction.Centrifugal slagging-off, ultrafiltration isolating protein and concentrated liquid glucose, after nanofiltration removed monose and inorganic salt, spraying drying or vacuum lyophilization prepared purified oligomerization acid solid preparation.
Application example
1. with the main component of the above-mentioned oligomerization acid that makes, can obviously promote the growth of plant as plant-growth regulator.
The germination that the OAS seed soaking of different concns is handled tomato seeds has promoter action, handles the germinating energy height of back tomato seeds, and it is more neat to germinate; The comparable contrast of germinating energy of the seed of handling through 10ppm OAS improves 11.34%.
OAS has promoter action to the growth of pea seedling, is in particular in that the number of blade increases, plant height increases, root increases, strain is heavy and root heavily increases; Compare with control group, the number of blade can increase 7.87%, plant height increases by 23.62%, root longly increases by 33.74%, strain heavily increases by 29.13%, root heavily increases by 47.62%; Root system vigor and photosynthetic pigments content also obviously improve.
2. with the main component of the above-mentioned oligomerization acid that makes, can reduce disease and pest, strengthen crop anti-adversity as biological pesticide.
At low temperature with damage to plants caused by sudden drop in temperature that oligomerization acid can improve 12.36%, 9.85%, 23.78% respectively with tomato seeds percentage of germination, germination index and activity index under the condition, reduce the injury of low temperature stress cell membrane, improve the activity of germinating seed, strengthen crop anti-adversity.
Claims (5)
1. bacterium is produced in an oligomerization acid, it is characterized in that: it belongs to brevibacterium sp, it is the bacterial strain that will obtain from the nature extraction separation, select culture medium culturing with sodium alginate, under 4 ℃ or normal temperature, preserve, the depositary institution that send of bacterial classification is Chinese typical culture collection center, and culture presevation day is on October 30th, 2007, and preserving number is CCTCC M 207168.
One kind according to claim 1 oligomerization acid produce the method that bacterium produces oligomerization acid, it is characterized in that: be the alginic-acid-decomposing bacteria enlarged culturing of CCTCC M 207168 with China's typical culture collection center preserving number earlier, prepare oligomerization acid with two-step approach again: the first step is used and is induced the product enzymic fermentation, preparation crude liquid type alginase preparation; The crude liquid type alginase preparation that second step used the first step to produce is degraded to sodium alginate soln, the acid of preparation oligomerization.
3. the production method of oligomerization acid according to claim 2, it is characterized in that: the expansion bacterial classification method of alginic-acid-decomposing bacteria is that the bacterial classification that will collect joins solid medium or liquid nutrient medium kind, temperature is 25-35 ℃, and the time is 1-3 days, obtains enlarging bacterial classification;
Described solid medium is that sodium alginate is selected substratum, wherein the weight percentage of each composition is: sodium alginate 0.5-2%, ammonium sulfate 0.5-1%, potassium primary phosphate or dipotassium hydrogen phosphate 0.05-0.1%, SODIUM PHOSPHATE, MONOBASIC or Sodium phosphate dibasic 0.05-0.1%, sal epsom, ferrous sulfate, each 0.01-0.1% of calcium chloride;
The weight percentage of described each composition of liquid nutrient medium is: sodium alginate 0.5-2%, urea 0.5-1%, potassium primary phosphate or dipotassium hydrogen phosphate 0.05-0.1%, SODIUM PHOSPHATE, MONOBASIC or Sodium phosphate dibasic 0.05-0.1%, sal epsom, ferrous sulfate, each 0.01-0.1% of calcium chloride; Or make solvent with seawater, and adding sodium alginate 0.5-2%, urea or other nitrogenous source 0.5-1%, and molysite, each 0.01-0.1% of calcium salt regulate the pH value to 6.0-8.0.
4. the production method of oligomerization acid according to claim 2, it is characterized in that: use and induce the product enzymic fermentation, the process of preparation crude liquid type alginase preparation is the bacterial classification that enlarged culturing obtains to be put into to be added with in advance induce in the fermentor tank that produces the enzymic fermentation substratum, at pH6.0-8.0, temperature 25-35 ℃ condition bottom fermentation 36-48 hour, the crude liquid type alginase preparation that the fermented liquid that obtains obtains after centrifugal slagging-off degerming;
Described inducing produced the enzyme substratum and comprised that sodium alginate selects substratum and add the 0.5-1% peptone or other nitrogen source and the short enzyme releasing hormone of 0.01-0.1%.
5. the production method of oligomerization acid according to claim 2, it is characterized in that: crude liquid type alginase preparation is degraded to sodium alginate soln, the process of preparation oligomerization acid is: the volume ratio 1 of pressing crude enzyme liquid and 2%-6% sodium alginate soln: 10-1: 20, mixing, at 40 ℃-50 ℃, carry out the enzyme digestion reaction of sodium alginate under the pH6.0-7.0 condition.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532042B (en) * | 2008-12-05 | 2011-10-05 | 王士奎 | Method for preparing oligomeric acid potassium or oligomeric acid ammonium and application thereof |
| CN103497260A (en) * | 2013-09-18 | 2014-01-08 | 农业部规划设计研究院 | Beta-oligomeric acid, and preparation method and application thereof |
| CN107417746A (en) * | 2017-08-08 | 2017-12-01 | 上海贯发海洋生物科技有限公司 | Oligomerization acid silicon compound and its preparation method and application |
| CN109295518A (en) * | 2018-09-29 | 2019-02-01 | 奚正华 | A kind of preparation method of flame-retardant modified spandex |
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2007
- 2007-11-16 CN CNA2007100505735A patent/CN101186892A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532042B (en) * | 2008-12-05 | 2011-10-05 | 王士奎 | Method for preparing oligomeric acid potassium or oligomeric acid ammonium and application thereof |
| CN103497260A (en) * | 2013-09-18 | 2014-01-08 | 农业部规划设计研究院 | Beta-oligomeric acid, and preparation method and application thereof |
| CN107417746A (en) * | 2017-08-08 | 2017-12-01 | 上海贯发海洋生物科技有限公司 | Oligomerization acid silicon compound and its preparation method and application |
| CN109295518A (en) * | 2018-09-29 | 2019-02-01 | 奚正华 | A kind of preparation method of flame-retardant modified spandex |
| CN109295518B (en) * | 2018-09-29 | 2021-07-27 | 东阳市华越针织有限公司 | Preparation method of flame-retardant modified spandex |
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Open date: 20080528 |