CN113637590A - Culture medium for culturing marine microalgae producing DHA (docosahexaenoic acid) grease as well as preparation method and application of culture medium - Google Patents

Culture medium for culturing marine microalgae producing DHA (docosahexaenoic acid) grease as well as preparation method and application of culture medium Download PDF

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CN113637590A
CN113637590A CN202110983639.6A CN202110983639A CN113637590A CN 113637590 A CN113637590 A CN 113637590A CN 202110983639 A CN202110983639 A CN 202110983639A CN 113637590 A CN113637590 A CN 113637590A
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adjust
starch
concentration
starch slurry
wheat
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赵祥颖
张华秋
刘建军
张家祥
刘丽萍
姚明静
田延军
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Shandong Food Ferment Industry Research & Design Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

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Abstract

The invention provides a culture medium for culturing marine microalgae producing DHA grease, and a preparation method and application thereof, and belongs to the technical field of deep processing of agricultural products. The wheat starch syrup prepared by the related process method contains rich nutrient components, and experiments prove that the wheat starch syrup is used as a culture medium raw material and is supplemented with corresponding auxiliary material components, so that the growth of schizophyllum can be effectively promoted, the lipid synthesis and DHA content of marine microalgae DHA can be effectively improved, artificial seawater is not needed any more, the production efficiency is greatly improved, the production cost is remarkably reduced, and the wheat starch syrup has good value in practical application.

Description

Culture medium for culturing marine microalgae producing DHA (docosahexaenoic acid) grease as well as preparation method and application of culture medium
Technical Field
The invention belongs to the technical field of deep processing of agricultural products, and particularly relates to a culture medium for culturing marine microalgae producing DHA grease, and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
DHA, docosahexaenoic acid, commonly known as "brain gold", is one of the polyunsaturated fatty acids essential for human body, and has important physiological functions of promoting brain development of human body, preventing and treating cardiovascular diseases, resisting inflammation and resisting cancer. At present, most of DHA products are derived from deep sea fish oil, DHA is extracted from the fish oil, the process is complex, the product yield is low, the fishy smell is still difficult to remove, and the application of the high value-added products in the food industry is greatly limited. The DHA content in the fish oil can obviously differ according to different fish species, fishing seasons, climates and places, the product quality fluctuation is large, and meanwhile, the marine ecology can be irreversibly damaged by over-fishing. Research shows that the DHA component in the deep sea fish oil is derived from marine microalgae. The DHA produced by the marine microalgae through the fermentation method is not limited by raw materials and production places, has no season and climate dependence, and can effectively reduce environmental hazards caused by over-fishing. Compared with fish oil products, the fish oil does not contain cholesterol and has no risk of pollution of heavy metals and the like. Therefore, the production of DHA grease by fermentation culture of marine microalgae is the direction of future industrial development. In 2011, the food additive docosahexaenoic acid oil (fermentation method) national food safety standard GB26400-2011 has been published in China, and the adopted marine microalgae for producing the (DHA) oil is recommended to be heterotrophic microalgae such as Schizochytrium sp, Ulkenia amoeboida or Crypthecodinium cohnii. The method is characterized in that microalgae is adopted to produce DHA grease by a fermentation method, an artificially synthesized culture medium is mainly adopted for cell culture, the composition of the culture medium has important influence on the concentration of cells in a culture solution and the accumulation of the intracellular DHA grease, the general microalgae cell culture at present mainly takes glucose as a main raw material, yeast extract, glutamic acid, ammonium salt and the like are generally required to be added as units and nutritional ingredients, and other inorganic ions are provided by artificial seawater. The raw material cost accounts for about 50-60% of the total production cost.
Wheat gluten production is one of the major deep processing modes of wheat, wherein the protein content of the wheat is about 12-13%, and the starch content is 65-70%, so that a large amount of wheat starch is co-produced in the process of producing wheat gluten. The wheat starch is divided into starch A and starch B according to the difference of particle sizes, the starch A has larger particles, is relatively easy to separate and refine, and can be further processed into commercial starch for sale. The B starch is starch with smaller particles, is usually mixed with non-starch components such as pentosan, residual protein and the like in wheat starch slurry in production and is difficult to separate, the starch component in the component contains about 80 percent of starch component in dry matters, and the B starch is mainly used for alcohol fermentation or sold as feed after being dried at present, and has lower added value of products.
Disclosure of Invention
Aiming at the problem of low-value utilization of wheat starch co-produced in the production process of wheat gluten powder at present, the invention provides a culture medium which is prepared by taking wheat starch syrup as a raw material and is used for culturing marine microalgae accumulating DHA oil. The wheat starch syrup prepared by the process method of the invention contains rich nutrient components, and experiments prove that the wheat starch syrup is used as a culture medium raw material and is supplemented with corresponding auxiliary material components, so that the growth of schizophyllum can be effectively promoted, the lipid synthesis and DHA content of marine microalgae DHA can be effectively improved, artificial seawater is not needed, and the wheat starch syrup has good practical application value.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
the invention provides an application of wheat starch slurry in preparing a culture medium for culturing DHA oil-producing marine microalgae.
In a second aspect of the invention, a culture medium for culturing marine microalgae producing DHA oil is provided, wherein the culture medium comprises wheat starch syrup, and the wheat starch syrup can be prepared from wheat starch slurry produced in a vital gluten production process.
Specifically, the wheat starch syrup is prepared by any one of the following methods (1) to (7):
(1) taking mixed starch slurry separated from wheat gluten, adding water to adjust the concentration of the starch slurry, adding liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, carrying out centrifugal separation after liquefaction, adding saccharifying enzyme to carry out saccharification after the pH of centrifugal supernatant is adjusted, and adding water to adjust the glucose concentration after the saccharification is finished to obtain wheat starch syrup;
(2) taking mixed starch slurry separated from wheat gluten, centrifuging, collecting supernatant liquid separately, adding water again to a precipitate part to adjust the concentration of the starch slurry, adding liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, carrying out centrifugal separation after liquefaction, processing the separated solid precipitate part separately, adding saccharifying enzyme to the centrifugal supernatant liquid after adjusting the pH value, carrying out saccharification, and adding water to adjust the glucose concentration after the saccharification is finished to obtain wheat starch syrup;
(3) taking mixed starch slurry separated from wheat gluten, centrifuging, collecting supernatant, adding a proper amount of crystalline glucose, and dissolving to obtain wheat starch syrup;
(4) taking wheat gluten to process and produce separated A starch slurry, adding water to adjust the concentration of the starch slurry, carrying out liquefaction and saccharification according to a conventional double-enzyme sugar preparation process, and adding water to adjust the concentration of glucose after saccharification is finished to obtain wheat starch syrup;
(5) taking wheat gluten to process and produce separated A starch slurry, centrifuging, separating clear liquid, adding water again to the precipitate part to adjust the concentration of the starch slurry, liquefying and saccharifying according to the conventional double-enzyme sugar preparation process, and adding water to adjust the concentration of glucose after saccharification is finished to obtain wheat starch syrup;
(6) taking wheat gluten to process and produce separated B starch slurry, adding water to adjust the concentration of the starch slurry, adding liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, adding saccharifying enzyme to carry out saccharification after regulating the pH value of centrifugal supernatant, and adding water to adjust the concentration of glucose after the saccharification is finished to obtain wheat starch syrup;
(7) and (2) taking the starch slurry B separated in the gluten processing production, centrifuging, separating clear liquid, adding water again to the precipitate part to adjust the concentration of the starch slurry, adding liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, carrying out centrifugal separation after liquefaction, adding saccharifying enzyme to the centrifugal supernatant after adjusting the pH, and adding water to adjust the glucose concentration after saccharification to obtain the wheat starch syrup.
In a third aspect of the present invention, there is provided a method for preparing the culture substrate, comprising: adding adjuvants into the wheat starch syrup.
In a fourth aspect of the present invention, there is provided a culture method of a marine microalgae producing DHA, the culture method comprising: and (3) inoculating the marine microalgae producing the DHA into the sterilized culture medium for fermentation culture.
The inoculation amount of the DHA-produced marine microalgae is controlled to be 5-10%; and (5) culturing until the residual glucose is reduced to below 0.1g/L, and finishing fermentation culture.
The DHA-producing marine microalgae include, but are not limited to, Scophyta nutans, Chlamydomonas wukenensis and Crypthecodinium cohnii, and more preferably Scophyta nutans.
The culture method also comprises the steps of collecting the DHA-producing marine microalgae obtained by fermentation culture, and separating to obtain DHA grease.
The beneficial technical effects of one or more technical schemes are as follows:
(1) the co-production of wheat starch syrup in the production process of wheat gluten contains starch and other nutrient components in wheat. Researches find that the glucose solution prepared from the wheat starch syrup is used as a raw material for microalgae cell culture, compared with the glucose solution which is used as the raw material, the addition of other nutrient substances can be reduced, the raw material cost is reduced, the cell concentration in fermentation liquor and the oil content in cells can be obviously improved, and the production efficiency is improved.
(2) Because the wheat starch contains non-starch components such as wheat gluten fiber, pentosan and the like, the saccharified sugar solution has high viscosity and difficult filtration, and the difficulty and the cost of separating and refining the product can be increased when the wheat starch is used as a fermentation raw material. The DHA grease is produced by the marine microalgae fermentation method, because the grease exists in cells, after the fermentation is finished, the cells can be firstly collected from the fermentation liquid by means of centrifugation and the like, and then the grease is separated, so that the extraction of the product is basically not influenced by the properties of the culture medium. Or the fermentation liquor can be directly used for providing DHA nutrition for animals in the breeding industry without cell collection. The wheat starch is used as the culture medium of the marine microalgae of DHA grease, the added value of the product is high, and the economic value of the wheat starch can be increased.
(3) The culture of the DHA-producing microalgae by using the wheat starch syrup as a matrix does not need artificial seawater, so that the corrosion of the artificial seawater to fermentation culture equipment is avoided.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
In a specific embodiment of the invention, the application of the wheat starch slurry in preparing the culture medium for culturing the DHA oil-producing marine microalgae is provided.
In another embodiment of the present invention, a culture medium for culturing marine microalgae producing DHA oil is provided, wherein the culture medium comprises wheat starch syrup, wherein the wheat starch syrup can be prepared from wheat starch produced in the production process of wheat gluten. According to the research of the invention, the wheat starch syrup is used as the culture medium of the DHA grease-producing marine microalgae, and only a proper amount of auxiliary materials are needed to be added for culture of the DHA grease-producing marine microalgae, so that the cell concentration of the DHA grease-producing marine microalgae in the fermentation liquor and the grease content in the cells can be obviously improved, the production efficiency is improved, and the raw material cost is greatly reduced.
In another embodiment of the present invention, the wheat starch syrup is prepared by any one of the following methods (1) to (7):
(1) taking mixed starch slurry separated from wheat gluten, adding water to adjust the concentration of the starch slurry, adding liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, carrying out centrifugal separation after liquefaction, adding saccharifying enzyme to carry out saccharification after the pH of centrifugal supernatant is adjusted, and adding water to adjust the glucose concentration after the saccharification is finished to obtain wheat starch syrup;
(2) taking mixed starch slurry separated from wheat gluten, centrifuging, adding water again to the precipitate part to adjust the concentration of the starch slurry, adding liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, centrifuging after liquefaction, adding saccharifying enzyme to carry out saccharification after the centrifugal supernatant is adjusted in pH, and adding water to adjust the concentration of glucose after saccharification is finished to obtain wheat starch syrup;
(3) centrifuging the mixed starch slurry separated from the wheat gluten to collect supernatant, and adding glucose to obtain wheat starch slurry;
(4) taking wheat gluten to process and produce separated A starch slurry, adding water to adjust the concentration of the starch slurry, carrying out liquefaction and saccharification according to a conventional double-enzyme sugar preparation process, and adding water to adjust the concentration of glucose after saccharification is finished to obtain wheat starch syrup;
(5) taking starch slurry A separated in the process of processing wheat gluten, centrifuging, adding water again to the precipitate part to adjust the concentration of the starch slurry, carrying out liquefaction and saccharification according to the conventional double-enzyme sugar preparation process, and adding water to adjust the concentration of glucose after the saccharification is finished to obtain wheat starch syrup;
(6) taking wheat gluten to process and produce separated B starch slurry, adding water to adjust the concentration of the starch slurry, adding liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, adding saccharifying enzyme to carry out saccharification after regulating the pH value of centrifugal supernatant, and adding water to adjust the concentration of glucose after the saccharification is finished to obtain wheat starch syrup;
(7) and (2) taking the starch slurry B separated in the gluten processing production, centrifuging, adding water again to the precipitate part to adjust the concentration of the starch slurry, adding liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, carrying out centrifugal separation after liquefaction, adding saccharifying enzyme to the centrifugal supernatant after adjusting the pH, and adding water to adjust the glucose concentration after the saccharification is finished to obtain the wheat starch syrup.
In another embodiment of the invention, in the method (1), water is added to adjust the concentration of the starch slurry to 10-15 Be, and the centrifugal supernatant is centrifuged to adjust the pH to 4.4-4.6; adding water to adjust glucose concentration to 10-15%; further preferably 10 to 12%; the solid precipitate fraction obtained by centrifugal separation after liquefaction can be processed separately, such as for the processing of wheat dietary fiber powder;
in another embodiment of the present invention, in the method (2), the specific conditions for the centrifugation of the mixed starch slurry are as follows: treating at 4500-5500rpm (preferably 5000rpm) for 5-10 min; adding water again to the precipitate part to adjust the concentration of the starch slurry to 10-15 Be; centrifuging the supernatant to adjust the pH to 4.4-4.6; adding water to adjust glucose concentration to 10-15%; further preferably 10 to 12%;
in another embodiment of the present invention, in the method (3), the specific conditions for the centrifugation of the mixed starch slurry are as follows: treating at 4500-5500rpm (preferably 5000rpm) for 5-10 min; adding glucose to adjust the concentration of glucose in the starch slurry to 10-15%; further preferably 10 to 12%;
in another specific embodiment of the invention, in the method (4), water is added to adjust the concentration of the starch slurry to 10-15 Be, and water is added to adjust the concentration of glucose to 10-15%; further preferably 10 to 12%;
in another embodiment of the present invention, in the method (5), the specific conditions for centrifuging the a-starch slurry are as follows: treating at 1500-; adding water again to the precipitate part to adjust the concentration of the starch slurry to 10-15 Be, and adding water to adjust the concentration of glucose to 10-15%, and further preferably 10-12%;
in another specific embodiment of the invention, in the method (6), water is added into the B starch slurry to adjust the concentration of the B starch slurry to 10-15 Be; centrifuging the supernatant to adjust the pH to 4.4-4.6; adding water to adjust glucose concentration to 10-15%, preferably 10-12%; separating the solid matrix in the liquefied liquid by a centrifugal method after liquefaction, wherein the solid matrix can be used for processing the wheat dietary fiber powder;
in another embodiment of the present invention, in the step (7), the specific conditions for centrifuging the B starch slurry are as follows: treating at 4500-5500rpm (preferably 5000rpm) for 5-10 min; adding water again to the precipitate part to adjust the concentration of the starch slurry to 10-15 Be; centrifuging the supernatant to adjust the pH to 4.4-4.6; adding water to adjust glucose concentration to 10-15%; further preferably 10 to 12%; separating the solid matrix in the liquefied liquid by a centrifugal method after liquefaction, wherein the solid matrix can be used for processing the wheat dietary fiber powder;
in another embodiment of the present invention, the culture medium further comprises an adjuvant, wherein the adjuvant comprises any one or more of sodium glutamate, ammonium sulfate and yeast extract, and the content of the sodium glutamate in the culture medium is 0-2%, preferably 1%;
in yet another embodiment of the invention, the ammonium sulfate is present in the culture medium in an amount of 0-0.5%, preferably 0.3%;
in still another embodiment of the present invention, the content of the yeast extract in the culture medium is 0 to 2%, preferably 0.5% or 1%.
In another embodiment of the present invention, there is provided a method for preparing the culture substrate, the method comprising: adding adjuvants into the wheat starch syrup.
In another embodiment of the present invention, there is provided a culture method of marine microalgae producing DHA, the culture method comprising: and (3) inoculating the DHA-producing marine microalgae into the sterilized culture medium for fermentation culture.
The inoculation amount of the DHA-produced marine microalgae is controlled to be 5-10%; and (5) culturing until the residual glucose is reduced to below 0.1g/L, and finishing fermentation culture.
The DHA-producing marine microalgae include, but are not limited to, Scophyta nutans, Chlamydomonas wukenensis and Crypthecodinium cohnii, and more preferably Scophyta nutans.
In another embodiment of the present invention, the cultivation method further comprises collecting the DHA-producing marine microalgae obtained by fermentation culture, and separating to obtain DHA oil.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
Experimental Material
Experimental strains: the strain is obtained from Schizosaccharomyces pombe of food fermentation industry research and design institute in Shandong province.
Preparing a wheat starch culture medium:
culture substrate i (ABC): taking mixed starch slurry separated from wheat gluten, adding water to adjust the concentration of the starch slurry to 10-15 Be, adding a proper amount of liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, centrifuging at 5000rpm for 3min after liquefaction, collecting supernatant, adding 1 time of water to a precipitate, washing for 1 time, centrifuging, and collecting supernatant. And combining the two supernatants, adjusting the pH value to 4.4-4.6, adding a proper amount of saccharifying enzyme for saccharification, adding water to adjust the glucose concentration to 10-12% after the saccharification is finished, and adding a proper amount of other required auxiliary materials to obtain the culture medium of the marine microalgae. The precipitate can be used for processing wheat dietary fiber powder.
Culture medium ii (AB): taking mixed starch slurry separated from wheat gluten, centrifuging at 5000rpm for 5min, collecting a precipitate, adding water again to adjust the concentration of the starch slurry to 10-15 Be, adding a proper amount of liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, centrifuging at 5000rpm for 3min after liquefaction, collecting a supernatant, adding 1 time of water to the precipitate, washing for 1 time, centrifuging, and collecting the supernatant. Mixing the two supernatants, adjusting pH to 4.4-4.6, adding appropriate amount of diastase, saccharifying, adding water to adjust glucose concentration to 10-12%, and adding appropriate amount of other adjuvants. The precipitate can be used for processing wheat dietary fiber powder.
Culture medium iii (a): taking mixed starch slurry separated from wheat gluten, centrifuging at 1500rpm for 1 second, and respectively collecting upper light phase and precipitate part, wherein the light phase part is starch slurry B, and the precipitate part is starch A. Adding water into the precipitate to adjust the concentration of the starch slurry to 10-15 Be, adding a proper amount of liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, adjusting the pH value to 4.4-4.6 after liquefaction, adding a proper amount of saccharifying enzyme to carry out saccharification, adding water to adjust the glucose concentration to 10-12% after the saccharification is finished, and adding a proper amount of other required auxiliary materials to obtain the starch slurry.
Culture substrate iv (AC): centrifuging the mixed starch slurry separated from the wheat gluten at 1500rpm for 1 second, and collecting the upper light phase and the precipitate part respectively, wherein the precipitate part is starch A. Adding water into the precipitate to adjust the concentration of the starch slurry to 10-15 Be, adding a proper amount of liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, adjusting the pH value to 4.4-4.6 after liquefaction, adding a proper amount of saccharifying enzyme to carry out saccharification, adding supernatant (C) of the starch slurry B, centrifuging at 5000rpm for 5min, adjusting the glucose concentration to 10-12%, and adding a proper amount of other required auxiliary materials to obtain the starch slurry.
Culture substrate v (BC): and (3) taking the starch slurry B, adding a proper amount of liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, centrifuging at 5000rpm for 3min after liquefaction, collecting supernatant, adding 1 time of water to a precipitate, washing for 1 time, centrifuging, and collecting supernatant. Mixing the two supernatants, adjusting pH to 4.4-4.6, adding saccharifying enzyme for saccharification, adding water to adjust glucose concentration to 10-12%, and adding appropriate amount of other adjuvants. The precipitate can be used for processing wheat dietary fiber powder.
Culture medium vi (B): and (3) centrifuging the starch slurry B at 5000rpm for 5min, collecting the precipitate, adding water again to adjust the concentration of the starch slurry to 10-15 Be, adding a proper amount of liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, centrifuging at 5000rpm for 3min after liquefaction, collecting the supernatant, adding 1 time of water to the precipitate, washing for 1 time, centrifuging, and collecting the supernatant. Mixing the two supernatants, adjusting pH to 4.4-4.6, adding appropriate amount of diastase, saccharifying, adding water to adjust glucose concentration to 10-12%, and adding appropriate amount of other adjuvants. The precipitate can be used for processing wheat dietary fiber powder.
Culture medium vii (C): centrifuging the mixed starch slurry separated from the wheat gluten at 5000rpm for 5min, collecting the supernatant, centrifuging to remove precipitate, and using the supernatant for preparing the culture medium.
Control culture medium (g/L): 120 parts of glucose, 10 parts of sodium glutamate, 3 parts of ammonium sulfate and 10 parts of yeast extract powder, and the yeast extract powder is prepared by artificial seawater diluted by 1 time.
Artificial seawater (g/L): NaCl 11.2, KCl 0.8, MgSO4 1.9，MgCl2 2.6，CaCl2 1.2，NaHCO30.2，NaBr 0.06。
Microalgae cell culture
The culture medium prepared by the method is prepared by adopting oral glucose in a contrast experiment. The culture medium is sterilized and inoculated into cultured schizochytrium SFD-1502 seed liquid, and fermentation culture is carried out at 28-30 ℃.
And (3) shake flask culture: the liquid loading capacity of a 500ml triangular flask is 50ml, the inoculation amount is 5-10%, and the culture is carried out for 3-4 days under the optimal culture condition.
Fermentation in a fermentation tank: the fermentation is carried out by adopting a 50L fermentation tank, the inoculation amount is 5-10%, and the proper temperature and the relative dissolved oxygen concentration are controlled according to requirements in the fermentation process. Culturing until the residual glucose is reduced to below 0.1g/L, and finishing fermentation.
Seed culture medium: 50g/L glucose, 10g/L yeast extract, 10g/L corn steep liquor, 5g/L sodium chloride, and pH 7.0.
Results of shaking flask experiments
Experiments show that the culture medium prepared by using the wheat starch has the effects on the growth of schizochytrium and the synthesis of DHA grease by using glucose as a control to perform a DHA fermentation production test on the prepared culture medium. The results are shown in Table 1:
TABLE 1 culture substrate culture DHA-producing Schizovibrio results
Figure BDA0003229831470000081
Figure BDA0003229831470000091
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. Application of wheat starch slurry in preparing culture medium for culturing marine microalgae producing DHA oil is provided.
2. A culture medium for culturing marine microalgae producing DHA oil is characterized by comprising wheat starch syrup, wherein the wheat starch syrup is prepared from wheat starch slurry produced in a vital gluten production process.
3. The culture medium according to claim 2, wherein the wheat starch slurry is prepared by any one of the following methods (1) to (7):
(1) taking mixed starch slurry separated from wheat gluten, adding water to adjust the concentration of the starch slurry, adding liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, carrying out centrifugal separation after liquefaction, adding saccharifying enzyme to carry out saccharification after the pH of centrifugal supernatant is adjusted, and adding water to adjust the glucose concentration after the saccharification is finished to obtain wheat starch syrup;
(2) taking mixed starch slurry separated from wheat gluten, centrifuging, adding water again to the precipitate part to adjust the concentration of the starch slurry, adding liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, centrifuging after liquefaction, adding saccharifying enzyme to carry out saccharification after the centrifugal supernatant is adjusted in pH, and adding water to adjust the concentration of glucose after saccharification is finished to obtain wheat starch syrup;
(3) centrifuging the mixed starch slurry obtained by separating the wheat gluten to collect supernatant, and adding glucose to obtain wheat starch syrup;
(4) taking wheat gluten to process and produce separated A starch slurry, adding water to adjust the concentration of the starch slurry, carrying out liquefaction and saccharification according to a conventional double-enzyme sugar preparation process, and adding water to adjust the concentration of glucose after saccharification is finished to obtain wheat starch syrup;
(5) taking starch slurry A separated in the process of processing wheat gluten, centrifuging, adding water again to the precipitate part to adjust the concentration of the starch slurry, carrying out liquefaction and saccharification according to the conventional double-enzyme sugar preparation process, and adding water to adjust the concentration of glucose after the saccharification is finished to obtain wheat starch syrup;
(6) taking wheat gluten to process and produce separated B starch slurry, adding water to adjust the concentration of the starch slurry, adding liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, adding saccharifying enzyme to carry out saccharification after regulating the pH value of centrifugal supernatant, and adding water to adjust the concentration of glucose after the saccharification is finished to obtain wheat starch syrup;
(7) and (2) taking the starch slurry B separated in the gluten processing production, centrifuging, adding water again to the precipitate part to adjust the concentration of the starch slurry, adding liquefying enzyme according to the starch content in the starch slurry to carry out conventional starch liquefaction, carrying out centrifugal separation after liquefaction, adding saccharifying enzyme to the centrifugal supernatant after adjusting the pH, and adding water to adjust the glucose concentration after the saccharification is finished to obtain the wheat starch syrup.
4. The culture substrate of claim 3,
in the method (1), water is added to adjust the concentration of the starch slurry to 10-15 Be, and the centrifugal supernatant is centrifuged to adjust the pH to 4.4-4.6; adding water to adjust glucose concentration to 10-15%; further preferably 10 to 12%; separately processing the solid precipitate obtained by centrifugal separation after liquefaction, including processing of wheat dietary fiber powder;
in the method (2), the specific centrifugal conditions of the mixed starch slurry are as follows: treating at 4500-5500rpm (preferably 5000rpm) for 5-10 min; adding water again to the precipitate part to adjust the concentration of the starch slurry to 10-15 Be; centrifuging the supernatant to adjust the pH to 4.4-4.6; adding water to adjust glucose concentration to 10-15%; further preferably 10 to 12%;
in the method (3), the specific centrifugal conditions of the mixed starch slurry are as follows: treating at 4500-5500rpm (preferably 5000rpm) for 5-10 min; adding glucose to adjust the concentration of glucose in the starch slurry to 10-15%; further preferably 10 to 12%;
in the method (4), water is added to adjust the concentration of the starch slurry to 10-15 Be, and water is added to adjust the concentration of glucose to 10-15%; further preferably 10 to 12%;
in the method (5), the specific centrifugation conditions of the starch slurry A are as follows: treating at 1500-; adding water again to the precipitate part to adjust the concentration of the starch slurry to 10-15 Be, and adding water to adjust the concentration of glucose to 10-15%, and further preferably 10-12%;
in the method (6), water is added into the starch slurry B to adjust the concentration of the starch slurry to 10-15 Be; centrifuging the supernatant to adjust the pH to 4.4-4.6; adding water to adjust glucose concentration to 10-15%, preferably 10-12%; separating the solid matrix in the liquefied liquid by a centrifugal method after liquefaction, wherein the solid matrix can be used for processing the wheat dietary fiber powder;
in the step (7), the specific centrifugation conditions of the starch slurry B are as follows: treating at 4500-5500rpm (preferably 5000rpm) for 5-10 min; adding water again to the precipitate part to adjust the concentration of the starch slurry to 10-15 Be; centrifuging the supernatant to adjust the pH to 4.4-4.6; adding water to adjust glucose concentration to 10-15%; further preferably 10 to 12%; after liquefaction, the solid matrix in the liquefied liquid is separated by a centrifugal method and is used for processing the wheat dietary fiber powder.
5. The culture medium of claim 3, further comprising an adjuvant comprising any one or more of sodium glutamate, ammonium sulfate and yeast extract.
6. The culture substrate of claim 5,
the content of the sodium glutamate in the culture medium is 0-2%, and the preferred content is 1%;
the content of the ammonium sulfate in the culture medium is 0-0.5%, preferably 0.3%;
the content of the yeast extract powder in the culture medium is 0-2%, preferably 0.5% or 1%.
7. A method for producing a culture substrate according to any one of claims 2 to 5, which comprises: adding adjuvants into the wheat starch syrup.
8. A culture method of DHA-producing marine microalgae is characterized by comprising the following steps: inoculating DHA-producing marine microalgae into the sterilized culture medium of any one of claims 2-5 for fermentation culture.
9. The cultivation method according to claim 8, wherein the DHA-producing marine microalgae inoculation amount is controlled to be 5-10%; and (5) culturing until the residual glucose is reduced to below 0.1g/L, and finishing fermentation culture.
10. The cultivation method according to claim 8, wherein the DHA-producing marine microalgae comprise Schizochytrium sp, Panicum gracile and Crypthecodinium cohnii, further preferably Schizochytrium sp;
the culture method also comprises the steps of collecting the DHA-producing marine microalgae obtained by fermentation culture, and separating to obtain DHA grease.
CN202110983639.6A 2021-08-25 2021-08-25 Culture medium for culturing marine microalgae producing DHA (docosahexaenoic acid) grease as well as preparation method and application of culture medium Withdrawn CN113637590A (en)

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