CN100503811C - Schizochytrium WZU4771, and application in preparing powder of DHA and grease - Google Patents
Schizochytrium WZU4771, and application in preparing powder of DHA and grease Download PDFInfo
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Abstract
This invention discloses Schizochytrium WZU477 and its application in preparing DHA powder and DHA grease. The Schizochytrium WZU477 is obtained by gathering rotten leaves and using pine pollen as the bait, and is preserved at China General Microbiological Culture Collection Center (CGMCC No. 1730). The Schizochytrium WZU477 can produce fermented liquid that has 42.5 g/L cell dry weight and 14.1 g/L DHA acid after cultured for 5 d under optimal conditions. The productivity of DHA is 2.76 g/L .d, twice that of previously reported DHA production.
Description
Technical field
The invention belongs to microbial technology field, specifically, relate to a kind of new schizochytrium limacinum (Schizochytrium) and application thereof.
Background technology
Docosahexenoic acid (cis-4,7,10,13,16,19-docosahexaenoic acid, be called for short DHA) be commonly called as DHA (docosahexaenoic acid), be brain, amphiblestroid important structure material, can also regulate central nervous system function, prevention and treatment cardiovascular disorder, diminish inflammation, suppress cancer.For a long time, the commercial source of DHA is fish oil and little algae.The output of fish oil and DHA content are all unstable, and extract is mingled with fishy smell.Little algae is cultivated needs illumination and carbonic acid gas, and production technique is numerous and diverse, the cost height.Along with the increase of population, the corresponding increase of DHA demand needs the big DHA of fine quality of searching amount newly to originate.Think that at present the candidate of potentialization is marine heterotrophic microorganism (comprising bacterium and eukaryotic microorganisms).The unsaturated fatty acid content of bacterium is less than eukaryotic microorganisms because unsaturated fatty acids only is present in the cytolemma of bacterium, and eukaryotic microorganisms more with the triglyceride level of unsaturated fatty acids as the energy storage material.
Thraustochytriale section (Thraustochytriaceae) is that a class is similar to little algae but lacks chloroplast(id) so eukaryotic microorganisms is given birth in not all right photosynthetic obligate sea, has a liking for corruption, extensively is present in the environment that organic solute is many and salinity is high.Think that in the past thraustochytriale section belongs to mycota (Fungi), chytrid door (Chytridiomycota), Oomycete (Oomycetes), Saprolegniales (Saprolegniales), and molecular biological result of study thinks that it more should belong to protobiont, now belong to pipe hair organic sphere (Stramenopila), do not wait hair door (Heterokonta), net Myxomycetes (Labyrinthulomycetes), thraustochytriale (Thraustochytriales) (Dick M W.Straminipilois fungi:Systematics of the peronosporomycetes, including accounts of the marinestraminipilous protistes, the plasmodiophorids, and similar organisms.Dordrecht:Kluwer Academic Publishers, 2001,269-287).
Thraustochytriale section is not pathogenic, do not produce poison, humanly directly ingests it by mussel in the food chain and clam, and U.S. FDA is classified it as generally recognized as safe level (GRAS, Generally Recognized as Safe).Genus thraustochytrium in the thraustochytriale section (Thraustochytrium) and schizochytrium limacinum belong to (Schizochytrium) be considered to DHA best microbe-derived (Ward O P, et al.Process Biochemistry, 2005,40:3628-3652).From mechanisms such as eighties of last century the nineties FAO and WHO be recommended in add DHA in baby, the junior food prescription since, thraustochytriale section produces DHA and is subjected to paying close attention to more widely.T.roseumATCC28210 cultivated 5 days in optimizing substratum, DHA productivity be 0.2 gram/(dying) (Singh A, etal.J Ind Microb, 1996,16:370-373).S.limacinum SR21 cultivated 5 days in the substratum that contains glucose (9%) or glycerine (12%), DHA productivity be 0.8 the gram/(dying) (Yokochi T, et al.Appl Microbiol Biotechnol, 1998,49:72-76).One strain may be that the thraustochytriale of S.mangrovei Raghu Kumar is carbon source with glucose, cultivated 107 hours, DHA productivity be 0.5 the gram/(dying) (Bowles R D, et al.J Biotechnol, 1999,70:193-202).When culture temperature is 25 ℃, a strain S.mangrovei cultivated in glucose-yeast extract medium 52 hours, DHA productivity be 1.27 gram/(dying) (Fan K W, et al.J IndMicrobiol Biotechnol, 2001,70:199-202).T.aureum HO cultivated 4 days in glucose (3%) substratum, DHA productivity be 1.55 the gram/(dying) (Huang Huiqin etc. the microorganism journal, 2002,42:498-501).T.roseum MF2 cultivated 4 days in glucose (4%) substratum, DHA productivity be 0.31 the gram/(dying) (Wu Kegang etc. Marine University Of Zhanjiang's journal, 2002,22 (4): 24-32).But the DHA productivity that present thraustochytriale is reached is not very high, and its industrialization is very restricted.
Summary of the invention
Technical problem to be solved by this invention provides a kind of schizochytrium limacinum (Schizochytrium) WZU4771, thereby improves DHA productivity, is beneficial to the industrialization of fermentative production DHA.
The rotted leaf that schizochytrium limacinum of the present invention (Schizochytrium) WZU4771 collects in the Oujiangkou Qidudao beach reed bed of Wenzhou City, Zhejiang Province adopts method of using pine pollen as the bait to separate and obtains, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 1st, 2006, it abbreviates CGMCC as, and deposit number is CGMCC No.1730.
Its concrete screening step is as follows:
1. primary dcreening operation substratum (grams per liter): glucose 10, yeast extract paste 1, peptone 1, agar 18 is prepared with seawater (taking from marine site, Wenzhou).Other adds Streptomycin sulphate and penicillin each 250 mg/litre and 3 mg/litre germanium dioxides;
2. sieve substratum (grams per liter) again: glucose 30, yeast extract paste 2, peptone 2 is prepared with seawater (taking from marine site, Wenzhou);
3. prescreening method: get the rotted leaf in coastal beach mangrove forest in Wenzhou and the reed bed, clean with the aseptic seawater that contains Streptomycin sulphate and penicillin, be seeded on the described primary dcreening operation culture medium flat plate, add an amount of aseptic seawater and aseptic pine tree pollen on flat board, cultivate a couple of days.Picking pine tree pollen is observed the culture that adheres to growth on pine tree pollen, the pine tree pollen that adheres to culture is transferred on the new flat board again cultivates, till obtaining pure growth;
4. multiple screen method: pure growth is inoculated into described the sieve again in the substratum, on shaking table, cultivates.Centrifugation obtains culture, is dried to constant weight, the weighing dry cell weight;
5. screening index: soxhlet extraction detects the lipid content in the dry cell weight, and vapor-phase chromatography detects the DHA content in the fat, is index with DHA productivity, and screening obtains the bacterial strain of high yield DHA.
Strain characteristic:
The formalness and the ultrastructure of observing the bacterial strain of described high yield DHA under scanning and perspective Electronic Speculum is characterized by: the nutrition thalline is wrapped in monokaryon for oval spherical by discontinuous cell walls.Cell walls is made of the fine and close scale which floor is pressed on together, at the distinguishable scale in the discontinuous place of cell walls, and the thick about 2-3 nanometer of scale.Form expolasm net radial, the root shape and be attached on the substrate, the expolasm net results from expolasm net form adult, and discontinuous director goes out from cell walls.The row vegetative propagation is converted into zoosporangium by the nutrition thalline, and many zoospores are arranged in it.In the zoospore generative process, the and then each nucleus division of cytokinesis is carried out, and forms tetraploid cell.The zoospore adnation not isometric two flagellums, and mastigoneme is arranged on forward direction flagellum both sides, and recurrent flagellum is shorter.
The cell walls that existence is made of scale, expolasm net form adult and expolasm net are the standards of existing evaluation thraustochytriale section, these characteristics can't under opticmicroscope, offer a clear explanation (Moss S T.The Biology ofMarine Fungi.Cambridge:Cambridge University Press, 1986).The zoospore of thraustochytriale forms mechanism two kinds, promptly interim division (the nucleus division is carried out cytokinesis after finishing fully) and continuity division (the and then each nucleus division of cytokinesis is carried out).The latter can form tetraploid cell.Form zoospore with continuity division, produce tetraploid cell, be schizochytrium limacinum feature (Alexopoulos C J, et al.Introductory Mycology (4th ed.) .New York:JohnWiley and Sons, 1996,743-750).
Therefore, the identification of strains of described high yield DHA is schizochytrium limacinum (Schizochytrium), called after WZU4771.Generally speaking, the thalline of genus thraustochytrium is bigger, may occur aborning assembling and conglomeration, and the growth velocity faster (Barclay W R.US Patent, 1997,5688500) that schizochytrium limacinum belongs to.
Described schizochytrium limacinum (Schizochytrium) WZU4771 is suitable for being grown in the ocean environment, and when the salinity and the pH value of substratum were similar to the ocean, cell growth and DHA accumulation were good.The also general microorganism of its optimal temperature is low, and optimum temps is 20-25 ℃.Can be that carbon source is carried out cell growth and DHA accumulation with glucose, maltose, sucrose and starch.Organic nitrogen source (peptone, yeast extract paste and corn steep liquor etc.) is more suitable for cell growth and DHA accumulation than inorganic nitrogen-sourced (urea, ammonium sulfate and SODIUMNITRATE etc.).
Described schizochytrium limacinum (Schizochytrium) WZU47711 is by optimizing substratum and culture condition, adopt traditional fermentation process, cultivated through 5 days, dry cell weight in the fermented liquid reaches that 42.5 grams per liters, DHA content reach 14.1 grams per liters, DHA productivity reaches 2.76 gram/(dying), exceeds 1 times nearly than reported bacterial strain.
Fermentation technique:
The optimization substratum of described schizochytrium limacinum (Schizochytrium) WZU47711 is to contain the carbon source of 5%-12% and the nitrogenous source of 0.5%-1.8%, and concentration of seawater is 25%-100%, and the pH value is 3.5-7.5; The inoculum size of optimizing culture condition and being with 3%-10% inserts described optimization substratum, under the condition of 25-35 ℃, 100-400 rev/min, adopts traditional fermentation process, cultivates 4-8 days.
Fermented liquid obtained somatic cells with 3000-8000 rev/min of centrifugal 10-30 minute.
Described schizochytrium limacinum (Schizochytrium) the WZU4771 somatic cells that adopts traditional fermentation process, obtained in described optimization substratum, with described optimization culture condition contains the grease of 35%-76% and the DHA of 18%-32% (all being benchmark with the dry cell weight), the highest dry cell weight reaches that 42.5 grams per liters, DHA content reach 14.1 grams per liters, DHA productivity reaches 2.76 gram/(dying), almost exceeds 1 times than reported bacterial strain.Gas-chromatography shows that the long chain polyunsaturated fatty acids spectrum of this bacterium is simple, except that DHA, only contains a spot of clupanodonic acid (cis-7,10,13,16,19-docosapentaenoic acid, abbreviation DPA), helps separation and the purifying of DHA.
Another technical problem to be solved by this invention provides the application of schizochytrium limacinum (Schizochytrium) WZU4771 in preparation DHA powder and DHA grease.
The production of DHA powder:
Described schizochytrium limacinum (Schizochytrium) WZU47711 somatic cells adopts traditional food drying technology, thalline moisture is reduced to be not higher than 12%.Add antioxidant, anti-caking agent and weighting agent, mixing obtains being applied to the DHA powder of food or feed.
Described DHA powder is the meal of yellow or light orange, its DHA content is not less than 6%, peroxide value is not higher than 6.0 milliequivalent/kilograms, total plate count is not higher than 50000/gram, the coliform approximate number is not higher than 90/100 grams, and pathogenic bacterium (duodenum 12 road pathogenic bacterium and pathogenic coccus) must not detect.
The greasy production of DHA:
Described schizochytrium limacinum (Schizochytrium) WZU47711 somatic cells adopts traditional Vegetable oil lipoprotein production technology, through leaching, come unstuck, depickling, decolouring, deodorization, dewaxing and filtration, add antioxidant, mixing obtains being applied to the DHA grease of food.
Described DHA grease is the clarification of yellow or light orange, transparent oily liquids, and its DHA content is not less than 20%, and acid value is not higher than 1.0 milligrams of potassium hydroxide/gram, and peroxide value is not higher than 6.0 milliequivalent/kilograms, and normal hexane is not higher than 50 mg/kg.
The invention has the advantages that: adopt method of using pine pollen as the bait, on the rotted leaf of collecting in the Oujiangkou Qidudao beach reed bed of Wenzhou City, Zhejiang Province, separate obtaining schizochytrium limacinum (Schizochytrium) WZU4771 that a plant height produces DHA.This bacterium contains abundant grease and DHA.In the substratum of optimizing, culture condition to optimize adopts traditional fermentation process, cultivates through 5 days, dry cell weight in the fermented liquid reaches that 42.5 grams per liters, DHA content reach 14.1 grams per liters, DHA productivity reaches 2.76 gram/(dying), exceeds 1 times nearly than reported bacterial strain.And gas-chromatography shows that the long chain polyunsaturated fatty acids spectrum of this bacterium is simple, except that DHA, only contains a spot of DPA, helps separation and the purifying of DHA.Adopt traditional food drying technology and Vegetable oil lipoprotein production technology, schizochytrium limacinum (Schizochytrium) WZU4771 somatic cells is developed as DHA powder and the grease that is applied to food or feed.
The present invention is described in further detail below by embodiment.
Embodiment
Embodiment 1
Get the rotted leaf in the Oujiangkou Qidudao beach reed bed of Wenzhou City, Zhejiang Province, be cut into diameter and be about the fragment that 1.5 centimetres sequin or length are 1.5 centimetres, clean with the aseptic seawater that contains each 1 grams per liter of Streptomycin sulphate and penicillin, be seeded on the described primary dcreening operation culture medium flat plate, add about 5 milliliters of aseptic seawater and a small amount of aseptic pine tree pollen on flat board, cultivated 4-5 days down at 25 ℃.Picking pine tree pollen is observed the culture that adheres to growth on pine tree pollen, the pine tree pollen that adheres to culture is transferred on the new flat board again cultivates, till obtaining pure growth.
Pure growth is inoculated into is equipped with in 50 milliliters of described 250 milliliters of triangular flasks that sieve substratum again, cultivated 3 days on 150 rev/mins shaking table, culture temperature is 25 ℃.Culture with 3500 rev/mins centrifugal 10 minutes, centrifugal again after the washing, 105 ℃ of dryings 3 hours, the weighing dry cell weight.
Soxhlet extraction detects the lipid content in the dry cell weight, and vapor-phase chromatography detects the DHA content in the fat, is index with DHA productivity, and screening obtains the bacterial strain of high yield DHA.
Formalness and ultrastructure according to observe the bacterial strain of described high yield DHA under scanning and perspective Electronic Speculum are accredited as schizochytrium limacinum (Schizochytrium), called after WZU4771.
Described primary dcreening operation substratum contains (grams per liter): glucose 10, and yeast extract paste 1, peptone 1, agar 18 is prepared with seawater (taking from marine site, Wenzhou).Other adds Streptomycin sulphate and penicillin each 250 mg/litre and 3 mg/litre germanium dioxides.
The described substratum that sieves again contains (grams per liter): glucose 30, and yeast extract paste 2, peptone 2 is prepared with seawater (taking from marine site, Wenzhou).
Embodiment 2
Choose several different carbon sources respectively, concentration is 10%, and the peptone with 0.8% is a nitrogenous source, and concentration of seawater is 50%, and the pH value is 6.Inoculum size is 10%, and culture temperature is 25 ℃, and shaking speed is 150 rev/mins, carries out the DHA fermentation of schizochytrium limacinum (Schizochytrium) WZU4771.Cultivated 5 days, the result is as shown in table 1, and glucose is optimum carbon source.
Table 1 carbon source is to the influence of DHA productivity
Embodiment 3
Choose several different nitrogenous sources respectively, concentration is 0.8%, and the glucose with 10% is carbon source, and concentration of seawater is 50%, and the pH value is 6.Inoculum size is 10%, and culture temperature is 25 ℃, and shaking speed is 150 rev/mins, carries out the DHA fermentation of schizochytrium limacinum (Schizochytrium) WZU4771.Cultivated 5 days, the result is as shown in table 2, and peptone is an optimum nitrogen source.
Table 2 nitrogenous source is to the influence of DHA productivity
Embodiment 4
Substratum contains 10% glucose and 0.8% peptone, and with the seawater preparation of different concentration of seawater, the pH value is 6 respectively.Inoculum size is 10%, and culture temperature is 25 ℃, and shaking speed is 150 rev/mins, carries out the DHA fermentation of schizochytrium limacinum (Schizochytrium) WZU4771.Cultivated 5 days, the result is as shown in table 3, the concentration of seawater optimum of 50%-100%.
Table 3 concentration of seawater is to the influence of DHA productivity
Embodiment 5
Substratum contains 10% glucose and 0.8% peptone, and concentration of seawater is 50%, regulates its pH value with hydrochloric acid or sodium hydroxide.Inoculum size is 10%, and culture temperature is 25 ℃, and shaking speed is 150 rev/mins, carries out the DHA fermentation of schizochytrium limacinum (Schizochytrium) WZU4771.Cultivated 5 days, the result is as shown in table 4, and optimal ph is 6.
Table 4 pH value is to the influence of DHA productivity
Embodiment 6
Substratum contains 10% glucose and 0.8% peptone, and concentration of seawater is 50%, and the pH value is 6.Inoculum size is 10%, and shaking speed is 150 rev/mins, under different culture temperature, carries out the DHA fermentation of schizochytrium limacinum (Schizochytrium) WZU4771.Cultivated 5 days, the result is as shown in table 5, and optimum temps is 20-25 ℃.
Table 5 temperature is to the influence of DHA productivity
Embodiment 7
Schizochytrium limacinum (Schizochytrium) WZU47711 somatic cells is dried to moisture at 105 ℃ and is not higher than 12%.Add antioxidant, anti-caking agent and weighting agent, mixing obtains being applied to the DHA powder of food or feed.
Embodiment 8
Schizochytrium limacinum (Schizochytrium) WZU4771 somatic cells is dried to moisture at 105 ℃ and is lower than 12%, leaches with hexane, and leaching progression is 4, and first three grade adopts the gradually rare mixing oil of concentration, and last step adopts fresh solvent, and total solvent ratio is 1: 1.
Hybrid oil filter is removed impurity such as solid suspension dregs of rice end, through 2 evaporations and No. 1 stripping, divides the hexane of leaving away under normal pressure, obtains crude oil.
Crude oil adds concentration and is 90% sulphuric acid soln (addition be crude oil amount 1%) under intensive stirs, add hot water (addition be crude oil amount 4%) again.Standing sedimentation takes out the upper strata crude oil, is neutral with hot water repetitive scrubbing to pH value.
Crude oil is under intensive stirs, and adding concentration is 8% sodium hydroxide solution (addition is determined excess 0.01% according to the acid number of crude oil).Be heated to 90 ℃, standing sedimentation is removed soap stock.
Crude oil is with 90 ℃ deionized water wash (the washing water yield be crude oil amount 50%), vacuum-drying then, and drying temperature is 100 ℃-105 ℃, vacuum tightness is 98.6 kPas.
Crude oil carries out adsorption bleaching (atlapulgite addition be crude oil amount 2.5%) with atlapulgite, and bleaching temperature is 90 ℃, and bleaching time is 20 minutes.Remove by filter atlapulgite.
The deodorization of crude oil water steam stripped, deodorization temperature is 180 ℃, and deodorization time is 2 hours, and steam consumption is 4% of a crude oil amount.
The decolouring crude oil fully is cooled to 25 ℃ under stirring at a slow speed, crystallization time is 48 hours, removes by filter wax.
Add antioxidant, obtain being rich in the finished product grease of DHA.
Claims (3)
1, schizochytrium limacinum (Schizochytrium) WZU4771 CGMCC No.1730.
2, the application of the described schizochytrium limacinum of claim 1 (Schizochytrium) WZU4771 in preparation docosahexenoic acid powder.
3, the application of the described schizochytrium limacinum of claim 1 (Schizochytrium) WZU4771 in the preparation docosahexaenoic acid grease.
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| CN102333880A (en) * | 2009-02-25 | 2012-01-25 | V.B.医疗私人有限公司 | Improved methods for fermentative production of docosahexaenoic acid |
| CN101812484B (en) * | 2009-03-20 | 2012-04-25 | 厦门汇盛生物有限公司 | Method for producing DHA by Schizochytrium in high-density culture through fermentation |
| CN101550078B (en) * | 2009-05-15 | 2012-02-22 | 淮海工学院 | Method of supercritical extraction of unsaturated fatty acid of schizochytrium limacinum |
| CN101575584B (en) * | 2009-06-18 | 2010-12-01 | 南京工业大学 | Vibrio schizogenum and method for producing DHA grease by using same |
| CN101892160B (en) * | 2010-01-06 | 2012-10-03 | 吉林省希玛生物科技有限公司 | Schizochytrium LX0809 (marine fungus) and industrial application thereof |
| CN101824442B (en) * | 2010-01-30 | 2012-06-20 | 中国海洋大学 | Fermentation production technique of schizogenesis chytrid |
| CN101914581B (en) * | 2010-07-27 | 2012-05-23 | 南京工业大学 | Method for improving fermentation yield of polyunsaturated fatty acid |
| CN102919512B (en) * | 2012-09-26 | 2015-05-27 | 内蒙古金达威药业有限公司 | DHA (docosahexaenoic acid)-rich microalgae powder and preparation method thereof |
| CN102864111B (en) * | 2012-10-10 | 2013-07-17 | 江南大学 | Schizochytrium limacinum strain for producing docosahexaenoic acid |
| CN104046568A (en) * | 2013-11-05 | 2014-09-17 | 北京大学深圳研究生院 | Culturing liquid rich in DHA Thraustochytriaceae, and culturing method |
| CN103981106B (en) * | 2014-06-03 | 2016-08-24 | 上海来益生物药物研究开发中心有限责任公司 | DHA superior strain and application thereof |
| CN114907988B (en) * | 2022-05-06 | 2024-06-11 | 江苏远大仙乐药业有限公司 | Schizochytrium limacinum, fermentation liquor and application thereof |
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Inventor after: Chen Shuirong Inventor after: Zhao Xiaowei Inventor after: Zhong Huichang Inventor after: Chen Liyi Inventor after: Zhou Maohong Inventor before: Zhao Xiaowei Inventor before: Zhou Maohong |
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Free format text: CORRECT: INVENTOR; FROM: ZHAO XIAOWEI ZHOU MAOHONG TO: CHEN SHUIRONG ZHAO XIAOWEI ZHONG HUICHANG CHEN LIYI ZHOU MAOHONG Free format text: CORRECT: ADDRESS; FROM: 325035 HIGHER EDUCATION PARK, CHASHAN, WENZHOU CITY, ZHEJIANG PROVINCE TO: 361000 NO.1337-1339, TONGJI MIDDLE ROAD, TONGJI INDUSTRIAL ZONE, XIAMEN CITY, FUJIAN PROVINCE |
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Effective date of registration: 20101213 Address after: 361000 No. 1337-1339, Central Road, Jilin Industrial Area, Fujian, Xiamen Patentee after: Xiamen Huisheng Biological Co., Ltd. Address before: 325035 Zhejiang province Chashan Wenzhou Higher Education Park Patentee before: Wenzhou University |