CN115058462A - Preparation method of DHA oil and DHA powder - Google Patents
Preparation method of DHA oil and DHA powder Download PDFInfo
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- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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Abstract
The application discloses a preparation method of DHA grease and DHA powder, which comprises the following steps: firstly, after amplification culture, inoculating a strain into a fermentation culture medium, performing three times of fermentation, and transferring to a tank after each time of fermentation to obtain fermentation liquor; adjusting the pH value of the fermentation liquor to be alkaline, heating, and performing enzymolysis wall breaking on the fermentation liquor by using complex enzyme to obtain enzymolysis liquid; centrifuging the enzymolysis liquid in a nitrogen environment, and dehydrating supernatant liquid to obtain DHA grease and fermentation filtrate; and concentrating, emulsifying, homogenizing and drying the fermentation filtrate to obtain DHA powder. The method for preparing the DHA grease and the DHA powder can reduce grease loss after wall breaking, improve oil yield and DHA content in the grease, obtain two products of the DHA grease and the DHA powder, and reduce environmental pollution.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a preparation method of DHA grease and DHA powder.
Background
DHA (docosahexaenoic acid), commonly known as NAOHUANG, is a polyunsaturated fatty acid which is very important to human body and belongs to an important member of Omega-3 unsaturated fatty acid family. DHA is not self-synthesized in the body and must be taken in through food. It has the functions of strengthening intelligence, promoting brain cell development and curing angiocardiopathy and cerebrovascular disease, etc., and is known as new-generation functional health-care factor, so that it can be extensively used in the infant food additive and medicine industry.
At present, DHA grease is mainly from fermentation production, and is extracted by a solvent after enzymatic wall breaking, but the process has high cost and large processing difficulty, and is easy to have subsequent problems of solvent residue or solvent pollution and the like. At present, DHA grease can also be obtained by enzymatic wall breaking and centrifugation, but the process can generate a large amount of fermentation filtrate along with the separation and extraction of the DHA grease, and a certain amount of DHA can remain in the fermentation filtrate; if the fermentation filtrate is directly discharged, not only is the residual DHA wasted, but also the protein, glucose, biological fiber, COD, BOD, and the like contained in the fermentation filtrate increase the cost of wastewater treatment.
Disclosure of Invention
In view of the above problems, the application provides a preparation method of DHA grease and DHA powder, which comprises the steps of performing amplification culture on a strain, inoculating the strain into a fermentation medium for three times of fermentation, adjusting the pH value of a fermentation broth to be alkaline, heating, performing enzymolysis wall breaking on the fermentation broth by using complex enzyme, centrifuging an enzymolysis liquid in a nitrogen environment, taking supernatant for dehydration treatment to obtain DHA grease and fermentation filtrate, and performing dehydration, emulsification and homogenization on the fermentation filtrate to obtain DHA powder. Solvent residue or solvent pollution caused by a solvent extraction process can be avoided, the preparation process of the DHA grease is simpler, and the oil yield and the DHA content in the grease are improved. Can also avoid DHA waste and environmental pollution caused by direct discharge of fermentation filtrate. And two products of DHA grease and DHA powder can be obtained simultaneously to be applied to different scenes.
In order to achieve the above purpose, the present application provides the following technical solutions:
a preparation method of DHA grease comprises the following steps:
step one, inoculating a strain into a fermentation medium after amplification culture;
step two, carrying out three times of fermentation, and carrying out tank transfer after each time of fermentation is finished to obtain fermentation liquor;
regulating the pH value of the fermentation liquor to be alkaline and heating;
step four, carrying out enzymolysis wall breaking on the fermentation liquor by using compound enzyme to obtain enzymolysis liquid;
step five, centrifuging the enzymolysis liquid in a nitrogen environment, and taking supernatant liquor;
step six, carrying out dehydration treatment on the supernatant to obtain DHA grease and fermentation filtrate;
and seventhly, concentrating, emulsifying, homogenizing and drying the fermentation filtrate to obtain DHA powder.
Preferably, the bacterial species include: any one of Schizochytrium, Chlamydomonas wukensonii, and Crypthecodinium cohnii.
Preferably, the fermentation medium comprises: glucose, yeast extract, corn dry powder and inorganic salt.
Preferably, in the second step, after each tank transfer, the fermentation medium is maintained at a preset concentration by means of replenishment.
Preferably, the preset concentration is 5-25%.
Preferably, in the second step, the first fermentation temperature is 35-40 ℃, the second fermentation temperature is 30-35 ℃, and the third fermentation temperature is 25-30 ℃.
Preferably, in the third step, the pH value of the fermentation liquor is adjusted to 7.5-9, and the temperature is increased to 50-60 ℃.
Preferably, the complex enzyme comprises: a mixture of at least two of cellulase, alkaline protease, pectinase, trypsin, and lipase.
Preferably, after the fourth step, the method further comprises: and step eight, grinding the enzymolysis liquid.
Preferably, after step six, the method further comprises: and step nine, adding pure water into the DHA grease for grinding, centrifuging in a nitrogen environment, and taking supernatant for dehydration treatment to obtain the DHA essential oil.
Firstly, taking a single DHA strain colony, inoculating the single DHA strain colony into a shake flask filled with a seed culture medium, and placing the shake flask in a shaking table for activating seeds to obtain a seed solution with higher activity. And performing amplification culture on the seed solution to obtain a secondary seed solution, and inoculating the secondary seed solution into a fermentation tank filled with a fermentation culture medium for fermentation culture. The strain used for fermentation is any one of Schizochytrium, Kloeckerella and Crypthecodinium cohnii. The seed culture medium comprises: glucose, yeast extract and inorganic salt mixture. The fermentation medium comprises: glucose, yeast extract, corn dry powder and inorganic salt mixture.
The fermentation process is carried out for three times, and the tank is transferred after each fermentation. The temperature of the third fermentation is gradually reduced: the first fermentation temperature is 35-40 ℃, the fermentation time is 2-3 days, the second fermentation temperature is 30-35 ℃, the fermentation time is 2-3 days, and the third fermentation temperature is 25-30 ℃, the fermentation time is 2-3 days. During fermentation, the concentration of the fermentation medium (glucose, yeast extract, corn dry powder and inorganic salt mixture) is always controlled to be 5-25%.
And obtaining fermentation liquor after the fermentation is finished. Adjusting the pH value of the fermentation liquor to 7.5-9, heating to 50-60 ℃, carrying out enzymolysis wall breaking by using a complex enzyme of a mixture of at least two of cellulase, alkaline protease, pectinase, trypsin and lipase, and carrying out enzymolysis for 4-6 h to obtain an enzymolysis liquid.
And centrifuging the enzymolysis liquid in a nitrogen environment, and dehydrating the supernatant to obtain DHA grease and fermentation filtrate. The centrifugal force is 5000-8000 g, and the separation factor is 3000-5000.
And concentrating, emulsifying, homogenizing and drying the fermentation filtrate to obtain DHA powder which can be used as a feed additive for livestock, aquaculture animals or companion animals.
In order to refine DHA oil, the enzymolysis liquid can be ground before centrifugation.
The DHA oil prepared by the process is crude oil and is used for further refining. Adding pure water into DHA oil, mixing, and grinding. Centrifuging the enzymolysis liquid in a nitrogen environment, taking supernatant liquid, and dehydrating to obtain DHA essential oil. The centrifugal force is 8000-12000 g, and the separation factor is 5000-6000.
The application provides a preparation method of DHA grease, which comprises the steps of inoculating a strain into a fermentation medium after amplification culture for three times of fermentation, adjusting the pH value of a fermentation liquid to be alkaline, heating, carrying out enzymolysis wall breaking on the fermentation liquid by using a complex enzyme, centrifuging enzymolysis liquid under a nitrogen environment, taking supernatant for dehydration treatment to obtain DHA grease and fermentation filtrate, dehydrating, emulsifying, homogenizing and drying the fermentation filtrate to obtain DHA powder, grinding and carrying out secondary centrifugation on crude oil to prepare DHA essential oil. Solvent residue or solvent pollution caused by a solvent extraction process can be avoided, the preparation process of the DHA grease and the DHA powder is simpler, and the oil yield and the DHA content in the grease are improved. Can also avoid DHA waste and environmental pollution caused by direct discharge of fermentation filtrate. And two products of DHA grease and DHA powder can be obtained simultaneously to be applied to different scenes.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the application. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:
fig. 1 is a flow chart of a preparation method of DHA oil of the present application;
fig. 2 is a flow chart of another DHA oil and fat preparation method of the present application.
Detailed Description
The technical solutions of the present application will be described clearly and completely below with reference to embodiments of the present application, and it should be apparent that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Referring to fig. 1, a method for producing DHA oil of the present application is described in examples 1 to 3.
Example 1
A preparation method of DHA grease comprises the following steps:
step 1, inoculating the schizochytrium limacinum into a fermentation medium after amplification culture. Wherein, the fermentation medium comprises the following components: 60g/L glucose, 10g/L yeast extract, 10g/L, NaCl15 dry corn powder, 15g/L KNO 3 2g/L。
Step 2, first fermentation: the temperature is 35 ℃, the fermentation is carried out for 3 days, the concentration of a fermentation medium is 5 percent, the fermentation is carried out in a tank, and the fermentation is carried out for the second time: fermenting for 2 days at the temperature of 30 ℃, wherein the concentration of a fermentation medium is 5 percent, transferring to a tank, and fermenting for the third time: fermenting at 25 deg.C for 3 days, and fermenting with 5% fermentation medium to obtain fermentation liquid.
And 3, adjusting the pH value of the fermentation liquor to 7.5 and heating to 50 ℃.
And 4, carrying out enzymolysis wall breaking on the fermentation liquor by using the complex enzyme for 6 hours to obtain enzymolysis liquid. Wherein the complex enzyme is a mixture of alkaline protease and lipase.
And 5, centrifuging the enzymolysis liquid in a nitrogen environment, and taking supernatant liquor. Wherein the centrifugal force is 5000g and the separation factor is 3000.
And 6, dehydrating the supernatant to obtain DHA grease and fermentation filtrate.
And 7, concentrating, emulsifying, homogenizing and drying the fermentation filtrate to obtain DHA powder.
Example 2
A preparation method of DHA grease comprises the following steps:
step 1, inoculating the incarcella wukense into a fermentation culture medium after amplification culture. Wherein, the fermentation medium comprises the following components: 60g/L glucose, 10g/L yeast extract, 10g/L, NaCl15 dry corn powder, 15g/L KNO 3 2g/L。
Step 2, first fermentation: fermenting for 2 days at 40 ℃, wherein the concentration of a fermentation medium is 10%, transferring to a tank, and fermenting for the second time: the temperature is 35 ℃, the fermentation is carried out for 3 days, the concentration of the fermentation medium is 10 percent, the fermentation is carried out in a tank, and the fermentation is carried out for the third time: fermenting at 30 deg.C for 2 days, and fermenting culture medium with concentration of 10% to obtain fermentation liquid.
And 3, adjusting the pH value of the fermentation liquor to 9 and heating to 50 ℃.
And 4, carrying out enzymolysis wall breaking on the fermentation liquor by using the complex enzyme for 4 hours to obtain enzymolysis liquid. Wherein the complex enzyme is a mixture of cellulase and pectinase.
And 5, centrifuging the enzymolysis liquid in a nitrogen environment, and taking supernatant liquor. Wherein the centrifugal force is 8000g and the separation factor is 5000.
And 6, dehydrating the supernatant to obtain DHA grease and fermentation filtrate.
And 7, concentrating, emulsifying, homogenizing and drying the fermentation filtrate to obtain DHA powder.
Example 3
A preparation method of DHA grease comprises the following steps:
step 1, performing amplification culture on Crypthecodinium cohnii, and then inoculating the Crypthecodinium cohnii into a fermentation culture medium. Wherein, the fermentation medium comprises the following components: 60g/L glucose, 10g/L yeast extract, 10g/L, NaCl15 g/15 g/L corn dry powder and KNO 3 2g/L。
Step 2, first fermentation: fermenting for 2 days at 40 ℃, wherein the concentration of a fermentation medium is 25%, transferring to a tank, and fermenting for the second time: the temperature is 30 ℃, the fermentation is carried out for 3 days, the concentration of the fermentation medium is 25 percent, the fermentation is carried out in a tank, and the fermentation is carried out for the third time: fermenting at 25 deg.C for 3 days, and fermenting culture medium with concentration of 25% to obtain fermentation liquid.
And 3, adjusting the pH value of the fermentation liquor to 8 and heating to 60 ℃.
And 4, carrying out enzymolysis wall breaking on the fermentation liquor by using the complex enzyme for 6 hours to obtain enzymolysis liquid. Wherein the complex enzyme is a mixture of cellulase, alkaline protease and trypsin.
And 5, centrifuging the enzymolysis liquid in a nitrogen environment, and taking supernatant liquor. Wherein the centrifugal force is 8000g and the separation factor is 3000.
And 6, dehydrating the supernatant to obtain DHA grease and fermentation filtrate.
And 7, concentrating, emulsifying, homogenizing and drying the fermentation filtrate to obtain DHA powder.
Example 4
A preparation method of DHA grease comprises the following steps:
step 1, inoculating the schizochytrium limacinum into a fermentation medium after amplification culture. Wherein, the fermentation medium comprises the following components: 60g/L glucose, 10g/L yeast extract, 10g/L, NaCl15 dry corn powder, 15g/L KNO 3 2g/L。
Step 2, first fermentation: fermenting for 2 days at 40 ℃, wherein the concentration of a fermentation medium is 15%, transferring to a tank, and fermenting for the second time: the temperature is 35 ℃, the fermentation is carried out for 2 days, the concentration of the fermentation medium is 15 percent, the fermentation is carried out in a tank, and the fermentation is carried out for the third time: fermenting at 25 deg.C for 3 days, and fermenting culture medium with concentration of 15% to obtain fermentation liquid.
And 3, adjusting the pH value of the fermentation liquor to 8.5 and heating to 60 ℃.
And 4, carrying out enzymolysis wall breaking on the fermentation liquor by using the complex enzyme for 5 hours to obtain an enzymolysis liquid. Wherein the complex enzyme is a mixture of alkaline protease and pectinase.
And 5, grinding the enzymatic hydrolysate.
And 6, centrifuging the enzymolysis liquid in a nitrogen environment, and taking supernatant liquor. Wherein the centrifugal force is 7000g and the separation factor is 4000.
And 7, dehydrating the supernatant to obtain DHA grease and fermentation filtrate.
And 8, concentrating, emulsifying, homogenizing and drying the fermentation filtrate to obtain DHA powder.
Example 5
A preparation method of DHA grease comprises the following steps:
step 1, inoculating the incarcella wukense into a fermentation medium after amplification culture. Wherein, the fermentation medium comprises the following components: 60g/L glucose, 10g/L yeast extract, 10g/L, NaCl15 dry corn powder, 15g/L KNO 3 2g/L。
Step 2, first fermentation: fermenting for 3 days at 37 ℃, wherein the concentration of a fermentation medium is 5%, transferring to a tank, and fermenting for the second time: fermenting for 3 days at the temperature of 32 ℃, wherein the concentration of a fermentation medium is 5 percent, transferring to a tank, and fermenting for the third time: fermenting at 30 deg.C for 3 days with 5% fermentation medium concentration to obtain fermentation liquid.
And 3, adjusting the pH value of the fermentation liquor to 7.5 and heating to 60 ℃.
And 4, carrying out enzymolysis wall breaking on the fermentation liquor by using the complex enzyme for 4 hours to obtain enzymolysis liquid. Wherein the complex enzyme is a mixture of trypsin and lipase.
And 5, centrifuging the enzymolysis liquid in a nitrogen environment, and taking supernatant liquor. Wherein the centrifugal force is 5000g and the separation factor is 3000.
And 6, dehydrating the supernatant to obtain DHA grease and fermentation filtrate.
And 7, concentrating, emulsifying, homogenizing and drying the fermentation filtrate to obtain DHA powder.
And step 8, adding pure water into the DHA grease for grinding, centrifuging in a nitrogen environment, and taking supernatant for dehydration treatment to obtain DHA essential oil. Wherein the centrifugal force is 8000g and the separation factor is 5000.
Example 6
A preparation method of DHA grease comprises the following steps:
step 1, performing amplification culture on Crypthecodinium cohnii, and inoculating the Crypthecodinium cohnii into a fermentation culture medium. Wherein, the fermentation medium comprises the following components: 60g/L glucose, 10g/L yeast extract, 10g/L, NaCl15 dry corn powder, 15g/L KNO 3 2g/L。
Step 2, first fermentation: fermenting for 3 days at 40 ℃, wherein the concentration of a fermentation medium is 25%, transferring to a tank, and fermenting for the second time: the temperature is 34 ℃, the fermentation is carried out for 3 days, the concentration of the fermentation medium is 25 percent, the fermentation is carried out in a tank, and the fermentation is carried out for the third time: fermenting at 28 deg.C for 2 days, and fermenting culture medium with concentration of 25% to obtain fermentation liquid.
And 3, adjusting the pH value of the fermentation liquor to 9 and heating to 60 ℃.
And 4, carrying out enzymolysis wall breaking on the fermentation liquor by using the complex enzyme for 4 hours to obtain enzymolysis liquid. Wherein the complex enzyme is a mixture of pectinase, trypsin and lipase.
And 5, centrifuging the enzymolysis liquid in a nitrogen environment, and taking supernatant liquor. Wherein the centrifugal force is 8000g and the separation factor is 5000.
And 6, dehydrating the supernatant to obtain DHA grease and fermentation filtrate.
And 7, concentrating, emulsifying, homogenizing and drying the fermentation filtrate to obtain DHA powder.
And 8, adding pure water into the DHA grease for grinding, centrifuging in a nitrogen environment, and taking supernatant for dehydration treatment to obtain the DHA essential oil. Wherein the centrifugal force is 12000g and the separation factor is 6000.
Referring to fig. 2, another method for producing DHA oil of the present application is described in example 7.
Example 7
A preparation method of DHA grease comprises the following steps:
step 1, taking schizochytrium limacinum to inoculate into a shake flask filled with a seed culture medium, and placing the schizochytrium limacinum in a shaking table to activate seeds to obtain a seed solution with high activity. And performing amplification culture on the seed solution to obtain a secondary seed solution, and inoculating the secondary seed solution into a fermentation tank filled with a fermentation culture medium. Wherein, the seed culture medium comprises the following components: 60g/L glucose, 10g/L, NaCl15 g/15 g/L yeast extract, KNO 3 2g/L，CaCO 3 8 g/L; the fermentation medium comprises the following components: 60g/L glucose, 10g/L yeast extract, 10g/L, NaCl15 dry corn powder, 15g/L KNO 3 2g/L。
Step 2, first fermentation: fermenting for 3 days at 40 ℃, wherein the concentration of a fermentation medium is 25%, transferring to a tank, and fermenting for the second time: the temperature is 35 ℃, the fermentation is carried out for 3 days, the concentration of the fermentation medium is 25 percent, the fermentation is carried out in a tank, and the fermentation is carried out for the third time: fermenting at 30 deg.C for 3 days with a fermentation medium concentration of 25% to obtain fermentation liquid.
And 3, adjusting the pH value of the fermentation liquor to 9 and heating to 60 ℃.
And 4, carrying out enzymolysis wall breaking on the fermentation liquor by using the complex enzyme for 6 hours to obtain enzymolysis liquid. Wherein the complex enzyme is a mixture of cellulase, alkaline protease, pectinase, trypsin and lipase.
And 5, grinding the enzymolysis liquid.
And 6, centrifuging the enzymolysis liquid in a nitrogen environment, and taking supernatant liquor. Wherein the centrifugal force is 8000g and the separation factor is 5000.
And 7, dehydrating the supernatant to obtain DHA grease and fermentation filtrate.
And 8, concentrating, emulsifying, homogenizing and drying the fermentation filtrate to obtain DHA powder.
And 9, adding pure water into the DHA grease for grinding, centrifuging in a nitrogen environment, and taking supernatant for dehydration treatment to obtain DHA essential oil. Wherein the centrifugal force is 12000g and the separation factor is 6000.
It should be noted that the above-mentioned embodiments illustrate rather than limit the application, and that those skilled in the art will be able to design alternative embodiments without departing from the scope of the appended claims.
Claims (10)
1. A preparation method of DHA grease and DHA powder is characterized by comprising the following steps:
step one, inoculating a strain into a fermentation medium after amplification culture;
step two, carrying out three times of fermentation, and carrying out tank transfer after each time of fermentation is finished to obtain fermentation liquor;
regulating the pH value of the fermentation liquor to be alkaline and heating;
performing enzymolysis and wall breaking on the fermentation liquor by using complex enzyme to obtain enzymolysis liquid;
fifthly, centrifuging the enzymolysis liquid in a nitrogen environment, and taking supernatant liquor;
step six, carrying out dehydration treatment on the supernatant to obtain DHA grease and fermentation filtrate;
and seventhly, concentrating, emulsifying, homogenizing and drying the fermentation filtrate to obtain DHA powder.
2. The method for preparing DHA oil and DHA powder according to claim 1, wherein the bacterial species includes: any one of Schizochytrium, Scytalidium wurkii and Crypthecodinium cohnii.
3. The method for producing DHA oil and DHA powder according to claim 1, wherein the fermentation medium includes: glucose, yeast extract, corn dry powder and inorganic salt.
4. The method for preparing DHA oil and DHA powder according to claim 1, wherein in the second step, the fermentation medium is maintained at a preset concentration by a supplementary method after each tank transfer.
5. The method for preparing DHA oil and DHA powder according to claim 4, wherein the preset concentration is 5-25%.
6. The method for producing DHA oil and DHA powder according to claim 1, wherein in the second step, the first fermentation temperature is 35 to 40 ℃, the second fermentation temperature is 30 to 35 ℃, and the third fermentation temperature is 25 to 30 ℃.
7. The method for preparing DHA oil and DHA powder according to claim 1, wherein in the third step, the pH value of the fermentation liquid is adjusted to 7.5-9, and the temperature is raised to 50-60 ℃.
8. The preparation method of DHA oil and DHA powder according to claim 1, wherein the complex enzyme comprises: a mixture of at least two of cellulase, alkaline protease, pectinase, trypsin and lipase.
9. The method for producing DHA oil and DHA powder according to claim 1, wherein after step four, the method further comprises:
and step eight, grinding the enzymolysis liquid.
10. The method for preparing DHA oil and DHA powder according to claim 1, wherein after the sixth step, the method further comprises:
and step nine, adding pure water into the DHA grease for grinding, centrifuging in a nitrogen environment, and taking supernatant for dehydration treatment to obtain DHA essential oil.
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| WO2024085425A1 (en) * | 2022-10-18 | 2024-04-25 | 씨제이제일제당 (주) | Method for producing microalgae concentrates with low viscosity |
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| WO2024085425A1 (en) * | 2022-10-18 | 2024-04-25 | 씨제이제일제당 (주) | Method for producing microalgae concentrates with low viscosity |
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