53 results about "Crypthecodinium cohnii" patented technology

The present invention relates to a process for preparing a biomass, such as from a microbial fermentation, for an extraction process to separate desired chemicals, nutritional products, bioactive components, proteins, carbohydrates, and lipids, from the biomass. Particularly preferred substances to extract include docosahexaenoic acid, docosapentaenoic acid, and arachidonic acid. The present invention also includes extracting the prepared biomass. Biomasses to be treated in accordance with the methods of the invention include plant, animal, and microbial biomass, particularly a microorganism such as Crypthecodinium cohnii and a fungus such as Mortierella alpina.

The present invention relates to a process for preparing a biomass, such as from a microbial fermentation, for an extraction process to separate desired chemicals, nutritional products, bioactive components, proteins, carbohydrates, and lipids, from the biomass. Particularly preferred substances to extract include docosahexaenoic acid, docosapentaenoic acid, and arachidonic acid. The present invention also includes extracting the prepared biomass. Biomasses to be treated in accordance with the methods of the invention include plant, animal, and microbial biomass, particularly a microorganism such as Crypthecodinium cohnii and a fungus such as Mortierella alpina.

The invention relates to a method for making docosahexaenic acid (DHA) grease through adopting bio-enzyme method wall breaking, comprising the following steps that: Crypthecodinium cohnii(Seligo)Javornicky is taken as starting strain; alga cell is obtained through carrying out continuous cultivation of Crypthecodinium cohnii(Seligo)Javornicky in liquid medium; and then DHA grease is extracted from the alga cell through bio-enzyme method wall breaking. The method has the advantages that: alga cell wall breaking is complete with the wall-breaking rate reaching more than 95 percent; the method does not need a drying step, and can prevent the oxidation of DHA crude oil, thereby ensuring that the peroxide value of extracted grease is extremely low (i.e. the POV value is less than 0.5); moreover, the yield of the extracted grease is more than 90 percent of total oil, while the refining yield is more than 70 percent of crude oil.

The present invention is process of fermenting Crypthecodinium cohnii(Seligo)Javornicky as one kind of marine micro algae to produce docosahexaenoic acid grease. The process includes continuous culturing Crypthecodinium cohnii(Seligo)Javornicky in liquid culture medium to obtain algae cell and extracting the algae cell to obtain docosahexaenoic acid grease. The present invention has proper marine micro algae strain, vitamins added into the culture medium and proper technological process, so that the present invention has high yield of docosahexaenoic acid grease, docosahexaenoic acid content in algae cell of 30-50 %, algae cell concentration of 20-40 g / L and oil content in algae cell of 20-50 %.

The invention discloses a DHA algae oil ethyl ester and a preparation method thereof. The method comprises the following steps: carrying out ethyl-esterifying on crypthecodinium cohnii DHA grease and then purifying trough a secondary molecular distillation method to obtain high-purity DHA ethyl ester. The invention further discloses a DHA glyceride and a method for transferring the DHA algae oil ethyl ester into glyceride. The method comprises the following steps: mixing the DHA algae oil ethyl ester with glycerinum in a material tank, feeding the mixed material in a reactor having immobilized lipase, performing transesterification to replace out the ethanol, recycling through a condenser, and flowing the reacted mixture back to the material tank, circularly reacting for 5-60 hours, and finally returning to the material tank, wherein the glyceride product is obtained by performing molecule distillation on the material. The method disclosed by the invention is suitable for the industrial production, the DHA greased produced from the microorganism is used as the raw material, the percent content of the DHA is greatly improved, the ethyl ester is transferred into the glyceride, the ethanol is avoided, the side effect after eating is not generated, and the utilization degree of the DHA algae oil ethyl ester in vivo is effectively improved.

The invention discloses a docosahexaenoic acid-producing bacterium and a mutation and screening method and application thereof. The strain is classified and named as Crypthecodinium cohnii HX-308, is preserved in China Center for Type Culture Collection, and has the preservation number M208246. The strain is a DHA-producing bacterium with high yield, has the DHA content 1.53 times of a starting strain, stable physiological-biochemical characteristic and stable heritability, and simultaneously can produce polyunsaturated fatty acid through liquid culture and fermentation; the content of unsaturated fatty acid reaches 20.4g / L, wherein the DHA content reaches 8.16g / L; and the strain is a good strain for producing natural DHA, and has excellent industrial production prospect.

The invention discloses a method for producing grease containing triglyceride type DHA by Crypthecodinium cohnii industrial fermentation. The method comprises the following steps: taking the Crypthecodinium cohnii as a strain, employing a culture medium comprising a carbon source, a nitrogen source and inorganic salts, and obtaining the DHA grease by fermentation; the nitrogen source contains hydrolytic liquor of slow-release bean pulp powder as nitrogen source. The grease produced by the fermentation contains more than 50% of DHA, among which EPA content is below 1%, and the DHA productivity reaches 4.6g / L per day. The DHA has high output, high purity, safe and reliable quality, low production cost, stable production, and little or no EPA; The method can produce environment-friendly and safe high DHA content triglyceride type DHA grease in quantity with low cost.

The invention discloses a method for extracting and preparing algae oil DHA, which comprises the following steps: (1) cultivating and fermenting Cryptidium koii; (2) drying; (3) pretreatment by breaking the wall; (4) microalgae DHA extraction; (5) urea inclusion enrichment and purification; the microalgae oil obtained in step (4) is enriched and purified by urea inclusion method to purify DHA; obtain microalgae DHA algae oil; (6) decolorization and deodorization; urea inclusion The microalgal DHA algae oil obtained by enrichment and purification is decolorized by adding activated clay accounting for 3% of the oil body mass. The decolorization temperature is 80°C and the decolorization time is 45 minutes. Steam stripping deodorization, the stripping temperature is 85°C, the vacuum degree is 0.30Kpa, the deodorization time is 1 hour, then slowly cool under the vacuum state, and centrifuge to obtain the algae oil DHA product.

The invention relates to a method for controlling microbe indexes in microalgae powder by combination of pasteurization and spray drying and a method for producing microalgae powder. The method comprises the following steps: pasteurizing a microalgae concentrated solution to obtain a sterilized concentrated solution, and drying the sterilized concentrated solution to obtain microalgae powder, thereby controlling the microbe indexes in the microalgae powder (including but not limited to total bacterial count, mold quantity and yeast quantity); and producing the microalgae powder. The method can effectively control the microbe indexes (especially total bacterial count) of the algae powder, and can maximally maintain the activity of the active substances in the microalgae. The method is suitable for but not limited to producing Chlorella, Haematococcus pluvialis, spirulina, Dunaliella, Nannochloropsis oculata, Schizochytrium, Crypthecodinium cohnii and various algae powders, and is hopeful to become a universal production method capable of effectively controlling microbe indexes (especially total bacterial count) in the algae powder.

The invention relates to a transformation method of Crypthecodinium cohnii. The method includes the following steps: pre-culturing Crypthecodinium cohnii; pre-culturing Agrobacterium; suspending the Crypthecodinium cohnii and Agrobacterium in the presence of an osmotic pressure regulator, and co-culturing complete Crypthecodinium cohnii cells and with Agrobacterium; and acquiring Agrobacterium transformed Crypthecodinium cohnii.

The invention relates to a method for controlling microbe indexes in microalgae powder by combination of microwave sterilization and spray drying and a method for producing microalgae powder. The method comprises the following steps: carrying out microwave sterilization on a microalgae concentrated solution to obtain a sterilized concentrated solution, and drying the sterilized concentrated solution to obtain microalgae powder, thereby controlling the microbe indexes in the microalgae powder (including but not limited to total bacterial count, mold quantity and yeast quantity); and producing the microalgae powder. The method can effectively control the microbe indexes (especially total bacterial count) of the algae powder, and can maximally maintain the biological activity of the active substances in the microalgae. The method is suitable for but not limited to producing Chlorella, Haematococcus pluvialis, spirulina, Dunaliella, Nannochloropsis oculata, Schizochytrium, Crypthecodinium cohnii and various algae powders, and is hopeful to become a universal production method capable of effectively controlling microbe indexes (especially total bacterial count) in the algae powder.

The invention discloses a method for mutagenizing micro algae to generate docosahexaenoic acid by use of ion beam bioengineering. The method comprises the following steps: 1) chemical mutagenesis: selecting crypthecodinium cohnii as the starting algae specie, performing slant culture according to a conventional method, culturing a seed inoculation bottle in a culture room at 25 DEG C, and diluting the micro algae in the seed liquid after about three days of culture with an artificial sea water solution; 2) ion beam mutagenesis: uniformly spreading the processed algae liquid in a sterile empty culture vessel, adding 20-30% glycerol for protection, drying with sterile air, placing a flat plate coated with micro algae into an ion implanter target room, and performing ion implantation; and 3) after the ion implantation is finished, screening unicellular algae by utilizing a solid medium, and picking the high-yield algae seeds from the shake flask in which the yield of docosahexaenoic acid cultured in the shake flask is 10%-20% higher than that of a control group. The method disclosed by the invention has the advantages of simple breeding method, relatively low cost and short breeding period.

The invention discloses a high-glucose-tolerance crypthecodinium cohnii domesticated strain, and a preparation method and an application thereof. The high-glucose-tolerance crypthecodinium cohnii strain is purchased from American Type Culture Collection ATCC, an algal strain with the code of ATCC 30556 is used as a starting strain, and crypthecodinium cohnii selects the strain from the ATCC (having the strain code of ATCC 30556) as the starting strain; the initial crypthecodinium cohnii ATCC 30556 is used as a processing object, and domestication culture is performed for multiple times in a by+ culture medium having glucose gradually added, and a processing object of each-time domestication culture is an algal strain obtained after last-time domestication culture; the glucose concentration is increased to 54 g / L after domestication is finished; the domestication process is performed totally for 650 days, and the total number of generations is 130; the obtained high-glucose-tolerance crypthecodinium cohnii domesticated strain is named as ALE3. The high-glucose-tolerance crypthecodinium cohnii domesticated strain has obvious economic advantages and industry prospects in fermentation production of docosahexaenoic acid (DHA).

The invention discloses a process for extracting and refining fatty acid rich in DHA, from crypthecodinium cohnii, which comprises that first crypthecodinium cohnii fermentation liquor is flocculated,then solid and liquid are separated, walls are broken via alkali, and cells are mechanically broken, then the broken thalluses are extractsed via organic solvent, to obtain DHA rough oil, and the DHArough oil is degelatinized, alkali refined, decolorized, and deodorized to obtain DHA fine oil. The invention has simple operation and reduced extraction cost of DHA fat, which only uses organic solvent in operation, eliminates residue finally, and improves DHA fat quality by refining, while the DHA content in extractive is 40-50%, with DHA batch production support.

The invention provides a method for producing DHA through Crypthecodinium cohnii fermentation. According to the method, Crypthecodinium cohnii is used as a strain and a culture medium including a carbon source, a nitrogen source, inorganic salt and trace elements is used, wherein 20% glycerol is added into the carbon source, DHA grease is prepared after three-stage fermentation, and in the fermentation process, a dissolved oxygen control strategy is adopted to control dissolved oxygen at 50%. In produced DHA fermentation liquid, the cell dry weight is 70-110 g / L, the grease content is 40-60%, the DHA content accounts for 45-55% of total grease, and the highest DHA producing rate is 8.7 g / (L.d) and is 1.89 times existing highest producing rate of DHA produced through the Crypthecodinium cohnii, which is reported so far. The technical indexes of the method provided by the invention are obviously better than the existing technical indexes; the produced DHA grease is high in yield, high in purity, low in cost and is beneficial to large-scale industrial production of the DHA.

The invention discloses a crypthecodinium cohnii mutant strain with high yield of DHA (docosahexaenoic acid), as well as a fermentation method and application thereof. The crypthecodinium cohnii mutant strain with the high yield of DHA, namely crypthecodinium cohnii 2.4K-2A2-52.4K-2A2-5 with CCTCCNO of M2012151 is screened out by 60Co-gamma ray radiation mutation of an ATCC30772 crypthecodinium cohnii strain. The grease quality and oil content of the strain are respectively improved by 51.66% and 66.37% in comparison with those of the original strain, the average DHA content achieves 39.04%, which is improved by 3.39% in comparison with that of the original strain, and the DHA yield which is calculated by g / L of a fermentation solution is improved by 56.92% in comparison with that of the original strain. The DHA produced by fermentation of the crypthecodinium cohnii ATCC30772 mutant strain has broad market prospects.

The invention discloses a method for preparing a fatty acid colloid solution applied to crypthecodinium cohnii for producing a docosahexaenoic acid. According to the method, the fatty acid colloid solution is artificially prepared and is applied to the heterotrophic culture of the crypthecodinium cohnii, so that the yield of a polyunsaturated fatty acid is obviously increased. The method comprises the steps that the ingredients of a fatty acid precursor are treated by adopting a specific ultrasonic emulsification method so as to form micro-nano granules dispersed into a culture solution, wherein the fatty acid stably exists in the solution and is not layered, accumulated or precipitated, and is supplied into a culture system; and after the crypthecodinium cohnii is continuously fermented for multiple days, the accumulation speed of cultured microalgae grease and the ratio of a long-chain fatty acid are obviously expedited and improved. The technical method is easy to implement and suitable for the artificial culture of heterotrophic microalgae.

The invention discloses a high-flux micro-fluidic chip for treating and collecting crypthecodinium cohnii. The chip is provided with an asymmetric contraction-expansion type micro-fluid channel. One side of the channel is connected with two liquid inlets, and the other side is connected with an outlet. One side of a sudden expansion cavity is connected with a liquid outlet. According to the high-throughput micro-fluidic chip for treating and collecting crypthecodinium cohnii, capture of a sample cell suspension is realized in the micro-channel, cells are kept to rotate in the micro-vortex according to a fixed track, crypthecodinium cohnii cells are treated by utilizing shear stress and are collected through a sheath flow outlet in one side of the cavity, the operation is simple, and the shearing precision is high; and meanwhile, the chip can be integrated with observation of the motion state of the crypthecodinium cohnii cells after partial structures of the crypthecodinium cohnii cells are sheared and DHA yield research, the morphological structure and the motion state of the crypthecodinium cohnii cells after the cells are under the action of shearing force in the vortex for a certain time are researched, and the DHA yield of the crypthecodinium cohnii cells is increased.

The invention discloses a method for producing grease containing triglyceride type DHA by Crypthecodinium cohnii industrial fermentation. The method comprises the following steps: taking the Crypthecodinium cohnii as a strain, employing a culture medium comprising a carbon source, a nitrogen source and inorganic salts, and obtaining the DHA grease by fermentation; the nitrogen source contains hydrolytic liquor of slow-release bean pulp powder as nitrogen source. The grease produced by the fermentation contains more than 50% of DHA, among which EPA content is below 1%, and the DHA productivityreaches 4.6g / L per day. The DHA has high output, high purity, safe and reliable quality, low production cost, stable production, and little or no EPA; The method can produce environment-friendly and safe high DHA content triglyceride type DHA grease in quantity with low cost.

The invention provides a method for improving heterotrophic microbial fermentation via multiple chemical promoters to produce docosahexenoic acid (DHA). The method comprises the following steps: with a heterotrophic culture medium of crypthecodinium cohnii as a foundation, adding any chemical promoter into the heterotrophic culture medium; the heterotrophic culture medium is composed of 9g / L of glucose, 2g / L of yeast extract powder and 25g / L of sea salt; the chemical promoter can also be called a plant growth regulator, including indoleacetic acid (IAA), naphthoxyessigsaeure (BNOA), chlorobenzoic acid, salicylic acid (SA), abscisic acid (ABA) and cholamine, and the concentrations of these chemical promoters in the heterotrophic culture medium are as follows: 2.5mg / L of indoleacetic acid (IAA), 4mg / L of naphthoxyessigsaeure (BNOA), 4mg / L of chlorobenzoic acid, 1mg / L of salicylic acid (SA), 20mg / L of abscisic acid (ABA) and 2.5mM cholamine; culturing at 25 DEG C for 72 hours, extracting grease, and measuring the DHA content of the culture by using a gas chromatograph-mass spectrometer, meanwhile, extracting cellular metabolite from a parallel culture, and detecting and analyzing the content change condition of the cellular metabolite by using a metabonomics method.

The invention belongs to microorganism fermentation, and particularly relates to a preparation method of crypthecodinium cohnii exopolysaccharides and application of the exopolysaccharides. The preparation method includes the process steps of crypthecodinium cohnii cultivation, exopolysaccharides concentrated solution obtaining and the like. The preparation method solves the technical problems that in the prior art, crypthecodinium cohnii exopolysaccharides are not effectively utilized and environment-friendly treatment of waste liquid generated by fermentation is complex. Crypthecodinium cohnii exopolysaccharides used by the method belong to the range of new resource food stated in China. Prepared crypthecodinium cohnii exopolysaccharides have good capacity for removing superoxide radicals, hydroxyl radicals and oxygen radicals, and good cell anti-oxidation vitality, have functional components corresponding to the anti-oxidation function, meet the requirements of corresponding specifications and standards of China, and can be effectively used for preparing antioxidant and making health care food with the anti-oxidation function.

The invention discloses a method for preparing high-yield natamycin by using crypthecodinium cohnii ATCC30772 fermented waste mushroom dregs. The method is used for adding the crypthecodinium cohnii ATCC30772 fermented waste mushroom dregs into a fermentation culture medium, thus improving the yield of natamycin. The method disclosed by the invention has the advantages of simple operation, low fermentation cost, energy conservation, environmental protection, relatively significantly-improved yield and the like.

The invention relates to a method for separating and purifying DHA (docosahexaenoic acid) from DHA oil produced by microbial fermentation. The method for separating and purifying DHA from DHA oil produced by microbial fermentation comprises the following steps: (1) taking the DHA oil, and carrying out esterification; (2) carrying out primary separation on esterified DHA oil by adopting a urea embedding-gradient freezing crystallization method; (3) carrying out molecular distillation on primarily separated fatty acid ethyl ester and then separating, so that high-purity DHA ethyl ester is prepared. The method for separating and purifying DHA from DHA oil produced by microbial fermentation is formed on the basis of coupling of multiple separation and purification means, DHA purification is obviously improved, DHA oil produced by crypthecodinium cohnii is taken as a raw material, and DHA ethyl ester with the purity more than 98% can be prepared; DHA oil produced by schizochytrium limacinum is taken as the raw material, and a product with the content of DHA ethyl ester and DPA ethyl ester more than 98% can be obtained.

The invention discloses a formula of a culturing agent for producing DNA from Crypthecodinium cohnii. The formula comprises, by mass, 7.2-8.8 parts of peptone, 1.8-2.2 parts of sodium nitrate, 16-22 parts of glucose, 4.5-5.5 parts of sodium acetate, 5.4-6.6 parts of sodium chloride, 1.44-1.76 parts of magnesium sulfate, 0.0054-0.0066 parts of VH, 0.0009-0.0011 parts of VB(12), and 900-1100 parts of water. Crypthecodinium cohnii is cultured according to the formula in order to realize high DHA output and large biomass.

The invention discloses an antibiotic namely hygromycin B that is sensitive to crypthecodinium cohnii producing DHA and a method using hygromycin B as the resistance screening marker to transform the gene of crypthecodinium cohnii. The method comprises the following steps: (1) determining the action concentration of hygromycin B; (2) establishing a gene transformation system; (3) preparing an exogenous gene section; (4) converting crypthecodinium cohnii by the exogenous gene section. According to the method, an exogenous gene can be introduced into cells of crypthecodinium cohnii, and at the same time, hygromycin B is taken as the screening marker to obtain an engineering strain of crypthecodinium cohnii with an exogenous gene. An important technological base is provided for the genetic engineering of crypthecodinium cohnii.

The invention relates to microbial fermentation, in particular to a mixed fermentation method for schizochytrium sp. and crypthecodinium cohnii. The method includes the steps that the crypthecodinium cohnii is inoculated in a culture medium containing a carbon source, a nitrogen source and essential nutrient elements to be fermented and prepared into fermentation liquor; when the crypthecodinium cohnii enters in a logarithmic phase, the schizochytrium sp. is inoculated into the fermentation liquor in the step A for conducting mixed fermentation; the crypthecodinium cohnii belongs to the crypthecodinium cohnii algae species capable of accumulating polyunsaturated fatty acids, and belongs to the crypthecodinium cohnii genus; the schizochytrium sp. belongs to the schizochytrium sp. algae species capable of accumulating polyunsaturated fatty acids, and the mass percentage composition of palmitic acid in the schizochytrium sp. is not lower than 30%. By the adoption of the method, the problems that when existing crypthecodinium cohnii and schizochytrium sp. are fermented independently, the technical defects of varying degrees exist, follow-up treatment is complex and the production cost is high are solved, and the advantages that the technological period is short, the production cost is low, prepared products can meet the related standard requirement without complex follow-up treatment and the quality of fat is high are achieved.

The invention belongs to the technical field of algae, and discloses a production, extraction and purification technology of DHA-enriched algae oil. The production, extraction and purification technology comprises the following steps of step (1) performing crypthecodinium cohnii seed culture; step (2) performing fermentation; step (3) performing extraction to obtain crude fat; and step (4) performing purification. Through adoption of the production technology disclosed by the invention, the biomass and the DHA yield of crypthecodinium cohnii can be increased; and besides, through the extraction and purification technology, the purify and the yield of the algae oil are increased, and the market application prospects are great.

The invention discloses a method of identifying DHA (docosahexaenoic acid) biosynthesis related fatty acid desaturase gene in crypthecodinium cohnii through De novo transcriptome sequencing, transcript splicing and differential expression analysis. The method particularly includes the following steps: (1) performing DHA fermentation culture to the crypthecodinium cohnii; (2) collecting thallus at different growth and DHA accumulation stages, and performing RNA extraction and transcriptome Illumina deep sequencing; (3) analyzing and pre-processing sequenced data; (4) performing function annotation and correlation statistic analysis to the spliced transcriptome, identifying the DHA biosynthesis related fatty acid desaturase in the crypthecodinium cohnii and acquiring a whole gene sequence thereof; and (5) by means of qRT-PCR analysis technology, performing expression verification to the DHA biosynthesis related fatty acid desaturase gene in the crypthecodinium cohnii. The method provides important approaches and knowledge basis for researching biosynthesis mechanism of DHA in the crypthecodinium cohnii and improving DHA synthesis capability of the crypthecodinium cohnii.

The invention discloses a biodiesel production method which comprises the following steps: inoculating crypthecodinium cohnii to a medium; irradiating for 10-14h per day with a red-blue double-color LED fill light; culturing for 6-7 days at 23-26 DEG C to obtain organic crypthecodinium cohnii cells; performing wall breaking treatment of the collected crypthecodinium cohnii cells, and extracting with an organic solvent to obtain microalgae oil; adding the microalgae oil, methanol and a zeolite molecular sieve catalyst into a reactor according to a mass ratio of 1:(5-7):(0.01-0.04) for an esterification reaction to obtain an esterified product; and separating and purifying the esterified product to obtain the biodiesel. In the method disclosed by the invention, by adopting the crypthecodinium cohnii with short production cycle, wide distribution, strong photosynthesis and high oil content as a raw material, the problems of vegetable oil raw material in biodiesel development are solved. The method also has the advantages of simple preparation technology, high catalysis efficiency, high oil extraction rate, high product yield and the like and lays a good foundation for the industrial and large-scale production of biodiesel.